ES2884155T3 - Serine protease variants and polynucleotides encoding the same - Google Patents

Abstract

A protease variant comprising a substitution at one or more positions corresponding to positions 1, 5, 9, 25, 29,30, 32, 38, 39, 40, 41, 42, 45, 46, 49, 50, 53 , 54, 55, 57, 59, 60, 61, 62, 64, 67, 68, 69, 74, 76, 77, 78, 79, 80, 81, 82, 87, 93, 94, 96, 99, 102 , 103, 105, 109, 111, 112, 114, 115, 117, 122, 123, 124, 128, 130, 136, 141, 142, 145, 146, 147, 150, 153, 154, 157, 159, 161 , 167, 169, 175, 182, 185, 199, 200, 205, 207, 209, 210, 213, 216, 217, 225, 228, 231, 234, 237, 242, 244, 245, 246, 248, 258 , 262, 266, 267, 269, 271, 272, 276, 278, 280, 284, 289, 293, 296, 299, 305, 308, 313, 314, 317, 318, 319, 321, 322, 324, 325 , 326, 329, 330, 331, 333, 336, 338, 341, 343, 345, 347, 348, 355, 358, 359, 361, 362, 363, and 364 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the polypeptide mature of SEQ ID NO: 3, and wherein the variant has increased thermostability as measured as enhancement factor, HIF, compared to the protease of SEQ ID NO: 3, wherein the variant comprises at least one substitution selected from the group that consists of : A1T, S5I, S5K, S5L, S5Y, T9H, K25P, S29I, S29P, S29R, S30E, K32W, F38L, 139G, I39L, I39V, I39Y, D40F, D40G, D40H, D40N, D40P, Q41I, Q41V , F42C, F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, A46E, K49P, S50C, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54I, Q54L, Q54M, Q54V, F55H, F55L, K57P , K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C, E74H, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A , E81H, E81K, E81L, E81P, E81Y, A82F, A82L, A82M, A82P, N87S, T93L, T93S, V94S, L96W, G99C, T102L, T102V, T103I, T103K, T103V, I105Q, D109H, D109M, D109M, D109M , F111H, F111K, F111S, F111Y, Q112R, G114L, N115A, N115C, N115K, N115P, N115R, N115T, N115Y, E117D, E117G, E117H, E117N, E117Q, E117S, I 122K, I122R, I123A, N124L, N124V, G128A, G128E, G128F, G128L, G128S, S130D, S130G, S130L, S130N, S130P, L136K, G141T, Q142A, Q142F, Q142L, Q142N, N145V, T146I, T146I, T146I, T146. T146N, I147S, K150F, K150L, K150R, N153A, N153L, N153M, N153P, N153R, N153Y, Q154A, Q154I, Q154L, Q154M, Q154N, Q154T, Q154V, N1571, N1571, N1571. D175I, D175N, Q182C, Q182T, Q182V, H185P, F199L, F199M, M200S, A205S, Q207H, Q207L, V209F, S210T, T213L, T213R, A216R, F217I, F217Y, V225Y, V225Y, I22. S237F, S237Q, A242I, A242L, A242V, G244S, S245P, T246S, S248Y, F258P, S262P, V266K, V266R, D267A, D267H, D267L, D267N, Q269L, V271R, S272I, S272K, S272L, S272R, S272T, S272V, S272Y, T276L, T276Y, G278P, D280N, D280S, D280T, D280Y, C284A, C284G, F289Y, I293T, V296A, V296I, R299N, K305S, L308V, P313L, F314I, F314I, F314I, F314I, F314I, F314I, S317 A319Q, K321L, K321Q, K321S, A322D, L324Q, L324S, N325L, D326P, S329C, S329H, S329I, S329L, S329N, S329T, S329V, S329W, G330Q, S331W, S331Y, P333R, S336K, S336N, N338M, N338Q, N338R, P341S, K343A, K343H, K343S, K343T, K343V, G345S, D347I, P348Y, P355I, P355R, A358L, A358P, A358R, A358Y, K359L, K359S, L361M, T362L, T362N, A363G, A363L, and V364I, and wherein substitution at the one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 1.4 .

Description

DESCRIPTION

Serine protease variants and polynucleotides encoding the same

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable format.

BACKGROUND OF THE INVENTION

The production of fermentation products, such as ethanol, from starch-containing material is well

known in the art. Two different types of procedures are generally used. The procedure more

commonly used, often referred to as a "conventional procedure", includes liquefying starch

gelatinized at high temperature typically using a bacterial alpha-amylase, followed by saccharification and

Simultaneous fermentation carried out in the presence of a glucoamylase and a fermenting organism. Other

well-known process, often referred to as a "crude starch hydrolysis" process

(RSH procedure) includes simultaneously saccharifying and fermenting granular starch below temperature

of initial gelatinization, typically in the presence of an acid fungal alpha-amylase and a glucoamylase.

US Patent No. 5,231,017-A describes the use of an acidic fungal protease during ethanol fermentation

in a process comprising liquifying gelatinized starch with an alpha-amylase.

Document WO 2003/066826 describes a crude starch hydrolysis process (RSH process) carried out

carried out in uncooked maceration in the presence of fungal glucoamylase, alpha-amylase and fungal protease.

WO 2007/145912 describes a process for producing ethanol comprising contacting

a suspension comprising granular starch obtained from plant material with an alpha-amylase capable of

solubilize granular starch at a pH of 3.5 to 7.0 and at a temperature below the gelatinization temperature

of starch over a period. from 5 minutes to 24 hours; obtain a substrate comprising more than 20% glucose,

and fermenting the substrate in the presence of a fermenting organism and enzymes that hydrolyze starch to a

temperature between 10°C and 40°C for a period of 10 hours to 250 hours. Additional enzymes added during

the contact step may include protease.

WO 2014/037438 describes wild-type serine proteases derived from Meripilus giganteus, Trametes

versicolor, and Dichomitus squalens and their use in animal feed. The T. versicolor protease shares

about 86% identity with the variant proteases of the invention.

WO 2015/197871 describes a mature amino acid sequence of a Meripilus protease

giganteus. This protease is identical to the wild-type parental protease used as a starting point in agreement

with the present invention to develop variant proteases having improved properties.

It is an object of the present invention to identify variants of the proteases of M. giganteus S53 that will give as

This results in increased storage stability (i.e., increased thermal stability) and improved performance.

Enhanced expression of the variant protease compared to the wild-type parental enzyme.

The present invention provides protease variants with improved properties compared to their parent.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to protease variants comprising a substitution in

one or more positions corresponding to positions 1,5,9,25,29,30,32,38,39,40,41,42,45,46,49,50,53,

54, 55, 57, 59, 60, 61, 62, 64, 67, 68, 69, 74, 76, 77, 78, 79, 80, 81, 82, 87, 93, 94, 96, 99, 102, 103, 105, 109, 111, 112, 114, 115, 117, 122, 123, 124, 128, 130, 136, 141, 142, 145, 146, 147, 150, 153, 154, 157, 159, 161, 167, 169,

175, 182, 185, 199, 200, 205, 207, 209, 210, 213, 216, 217, 225, 228, 231, 234, 237, 242, 244, 245, 246, 248, 258,

262, 266, 267, 269, 271, 272, 276, 278, 280, 284, 289, 293, 296, 299, 305, 308, 313, 314, 317, 318, 319, 321, 322,

324, 325, 326, 329, 330, 331, 333, 336, 338, 341, 343, 345, 347, 348, 355, 358, 359, 361, 362, 363, and 364

polypeptide of SEQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at

least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identity of the

sequence with the mature polypeptide of SEQ ID NO: 3, and wherein the variant has increased thermostability

measured as enhancement factor, HIF, compared to the protease of SEQ ID NO: 3, wherein the variant

comprises at least one substitution selected from the group consisting of: A1T, S5I, S5K, S5L, S5Y, T9H, K25P,

S29I, S29P, S29R, S30E, K32W, F38L, I39G, I39L, I39V, I39Y, D40F, D40G, D40H, D40N, D40P, Q41I, Q41V, F42C,

F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, A46E, K49P, S50C, S50I, S50L, S50M, S50Q, A53C, A53E, A53V,

Q54I, Q54L, Q54M, Q54V, F55H, F55L, K57P, K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G,

T64I, L67V, Q68V, Q68Y, T69C, E74H, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A, E81H, E81K, E81L,

E81P, E81Y, A82F, A82L, A82M, A82P, N87S, T93L, T93S, V94S, L96W, G99C, T102L, T102V, T103I, T103K, T103V,

I105Q, D109H, D109K, D109m, D109Y, F111H, F111K, F111S, F111Y, N115C, N115K, N115P, N115R, N115T, N115Y, E117D, E117G, E117H, E117N, E117Q, E117S, I122K, I122R, I123A, N124L, N124V, G128A, G128E, G128F, G128L, S130L, S130N, S130P, L136K, G141T, Q142A, Q142F, Q142L, Q142N, N145V, T146A, T146i, T146L, T146N, T146L T146N I147S, K150F, K150L, K150R, N153A, N153L, N153M, N153P, N153R, N153Y, Q154A, Q154I, Q154L, Q154M, Q154N, Q154T, Q154V, N157I, N157L, Y159H, Q167F E, T11661E,

I169S, D175I, D175N, Q182C, Q182T, Q182V, H185P, F199L, F19M, Q207L, V209F, S210T, T213L, T213R, A216R, F217i, F217Y, V225Y, I228L, I228T, I228W, Y231i, I228W, Y231i S234Q, S237F, S237Q, A242I, A242L, A242V, G244S, S245P, T246S, S248Y, F258P, S262P, V266K, V266R, D267A, D267H, D267L, D267N, Q269L,

V271 R, S272i, S272K, S272L, S272R, S272Y, T276L, T276Y, G278P, D280N, D280T, D280Y, C284A, C284G, F289Y, I293T, V296A, V296I, R299N, K305S, L308V, R299N, K305S, L308V, P313L , F314i, F314L, S317E, S317N, S317W, S318P, A319F, K321Q, K321S, A322D, L324Q, L324S, N325L, D326P, S329L S329N, S329T, S329V, S329W, G330Q , S331W, S331Y, P333R, S336K, S336N, N338M, N338Q, N338R, P341S, K343A, K343H, K343S, K343T, K343V, G345S, D347I, P348Y, P355I, P355R, A358L, A358P, A358R, A358Y, K359L, K359S , L361M, T362L, T362N, A363G, A363L, and V364I, and wherein substitution at one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least

1.4.

In a second aspect, the present invention relates to protease variants comprising a substitution at one or more positions corresponding to positions 20, 27, 29, 34, 40, 42, 45, 46, 50, 53, 54, 55 , 58, 59, 60, 61,

62, 66, 69, 76, 81,87, 93, 96, 98, 101, 102, 103, 109, 110, 115, 117, 122, 124, 125, 127, 128, 130, 136, 141, 142, 145,

146, 148, 149, 150, 153, 154, 157, 159, 161, 164, 167, 182, 183, 184, 185, 188, 199, 200, 207, 208, 209, 210, 211,

213, 216, 219, 228, 231, 237, 244, 249, 252, 253, 261, 266, 267, 272, 275, 276, 277, 280, 285, 294, 296, 299, 303, 304, 305, 306, 307, 308, 310, 317, 318, 319, 321, 326, 327, 336, 338, 341, 358, 359, 361,362, 363, and 364 of the polypeptide of SEQ ID NO: 3, and the variant has activity protease, and wherein the variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide mature of SEQ ID NO: 3, and wherein the variant has an increased specific activity measured as enhancement factor, AIF, compared to the protease of SEQ

ID NO: 3, wherein the variant comprises at least one substitution selected from the group consisting of: G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, D40S, D40Y, F42Y , K45L, K45Y, A46E, A46K, S50A, S50C, S50I, S50L, S50T, A53I, A53K, A53L, A53S, Q54L, F55R, D58M, I59M, I59P, S60C, S60E, S60P, S61D, S61P, S62C, S62N , S66F, S66L, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, E81S, N87F, T93S, L96F, L96S, L96T, T98L, P101F, P101L, P101M, P101R , T102C, T102I, T103V, D109P, D110W, N115A, N115D, E117F, E117G, E117L, E117W, E117Y, I122L, N124D, N124I, N124K, N124L, N124M, N124T , N124V, F125K, F125L, F125M, L127C, L127P, G128N, G128Y, S130D, S130F, S130I, S130N, L136C, G141A, G141C, G141F, G141L, G141M, G141Q, G141R, G141V, Q142A , Q142L, Q142M, Q142S, Q142W, N145F, N145G, N145I, N145L, N145P, N145R, N145S, N145V, N145W, T146A, T146C, T146H, T146I, T146M, T146N, T146Q, T146Q, T146W, S148G, A149V, K150F, N153D, N153F, N153G, Q154C, Q154F, Q154G, Q154L, Q154M, Q154L, Q154V, Q154T, Q154V, Q154W, N157F, N157A, N157E, N157F, N157A, N154E N157Y, Y159F, Y159G, Y159H, Y159S, Q161A, A164L, T167D, T167V, Q182F, S183A, S183F, A184F, A184H, A184I, A184K, A184L, A184Q, A184R, A184S, A184T, A184V, A184W, A184Y, H185A, H185L, H185P, H185S, N188L, N188Q, M200L, Q207F, G208H, V209F, V209G, V209W, V209Y, S210E, S210L, S210R, S210T S210Y, P211F, T213F, T213H, T213I, T213L, T213N, T213R, T213S, T213W, T213Y, A216F, A216L, S219M,

I228F, I228H, I228L, I228M, I228Q, I228V, I228Y, Y231F, Y231S, S237F, S237I, S237K, S237L, G244L, G244N, G244P, G244R, G249C, N252F, N252K, N252S, R253C, V261A, V261G, V266C, V266R, D267S, S272I, S272K, S272L, S272M, S272Q, S272Y, Q275A, Q275F, Q275R, Q275V, T276F, T276R, T276V, I277L, I277V, D280F, D280H, D280M, D280F, D280H, D280M, D280F D280S, D280T, A285L, A285S, T288C, T288I, T288V, S291V, V292C, I293M, I293Y, S294T, S294V, V296L, R299, A303C, A303F, A303H, A303S, A303V, G304H, G304K, G304P, G304R, G304S G304Y, K305C, K305F, K305H, K305i, K305L, K305M, S306L, S306M, S306R, P307C, L308P, L308T, F310M, F310P, F310Y, S317A, S317C, S317G, S317H, S317i, S317K S317L, S317R, S317W, S317Y, S318N, S318R, A319F, A319W, D326P, V327C, V327T, S336T, N338H, N338S, N338T, P3411, P341N, P341 R, A344K, A344L, A344P, G345K, G345S , P348G, P348L, P348S, N356L, N356Y, A358F, A358L, A358P, K359C, K359L, K359S, K359W, L361E, L361M, L361P, L361R, L361S, L3 61T, L361V, T362A, T362C, T362H, T362I, T362K, T362L, T362M, T362P, T362Q, T362R, T362V, T362Y, A363E, A363F, A363L, A363M, A363V, V364F, V364L, V364L, V364L, V364L, V364L, V364L wherein substitution at one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 1.37.

In a third aspect, the present invention relates to protease variants comprising a substitution at one or more positions corresponding to positions 2, 17, 22, 41,41,42, 45, 46, 54, 55, 57, 59 , 60, 61,62, 63, 64, 66,

69, 80, 82, 84, 87, 98, 99, 102, 103, 112, 114, 115, 129, 131, 134, 149, 159, 167, 169, 199, 200, 205, 207, 209, 211,

213, 217,242, 250, 266, 267, 269, 270, 271, 272, 274, 278, 279, 280, 284, 288, 291, 292, 294, 296, 301, 307, 314,

329, 331, 333, 336, 362, and 363 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at least 95%, at least 96%, at at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, and wherein the variant has an increased expression level measured as an enhancement factor , EIF, compared to the protease of SEQ ID

NO: 3, and wherein the variant comprises a substitution at one or more positions selected from the group consisting

In : I2L, A17P, P22V, Q41L, Q41T, Q41V, Q41Y, F42R, K45S, A46L, A46W, Q54R, Q54S, Q54V, Q54Y, F55L, F55N, F55T, F55Y, K57H, K57L, K57V, I59D, I59K, I59R, I59S, I59V, S60I, S60L, S60Q, S60R, S60Y, S61A, S61F, S62H, S62I, S62L, S62R, S62V, T63A, T63E, T63G, T63R, T63V, T64C, T64D, T64M, S66G, T69A, T69F, T69P, T69R, T69Y, S80G, S80L, S80N, S80T, A82H, I84G, N87P, T98C, G99V, T102A, T102H, T103F, T103I, T103V, Q112H, Q112M, Q112R, Q112S, G114LC, G114LC, G114LC, G114LC, G114LC, G114P, N115Y, E129L, E129S, E129V, E129Y, N131I, Q134A, Q134L, Q134R, A149D, Y159F, Y159L, T167A, T167L, T167S, T167W, I169S, F199Y, M200L, A205S, Q207N, V209P, P211 F , I270L, I270S, V271F, V2711, V271K, V271M, S272A, S272N, G274R, G278S, V279i, D280Y, C284A, T288A, T288C, T288G, S291V, V292S, S294G, V296P, V296R, V296T, I301T , P307T, F314L, S329D, S329V, S331A, S331G, S331T, P333V, S336G, S336L, S336T, T362R, and A363T, and wherein substitution at one or more positions provides a protease variant that has increased expression level in Aspergillus oryzae measured as enhancement factor, EIF, of at least 1.38.

The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods for producing the variants. In a further aspect, the present invention relates to compositions comprising the variants of the invention.

The present invention also relates to a process for preparing a fermentation product from starch-containing material, comprising simultaneously saccharifying and fermenting starch-containing material using enzymes that generate a carbohydrate source and a fermenting organism at a temperature below the initial gelatinization temperature of said starch-containing material in the presence of a variant protease. In another aspect, the present invention relates to a process for preparing a fermentation product from starch-containing material, comprising the steps of:

(a) liquifying starch-containing material in the presence of an alpha-amylase;

(b) saccharifying the liquefied material obtained in step (a) using a glucoamylase;

(c) fermenting using a fermenting organism;

wherein a variant protease of the invention is present during step b) and/or c).

DEFINITIONS

Protease: The term "protease" (also referred to as peptidases, proteinases, peptide hydrolases, or proteolytic enzymes) means a proteolytic activity (EC 3.4) that catalyzes the cleavage of peptide bonds. For purposes of the present invention, serine protease activity is determined according to the procedure described in the Examples. In one aspect, variants of the present invention have at least 20%, e.g. e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the protease activity of the mature polypeptide of SEQ ID NO: 2.

Protease activity: The term "protease activity" means proteolytic activity (EC 3.4). There are several types of protease activity, such as trypsin-like proteases that cleave at the carboxy-terminal side of Arg and Lys residues, and chymotrypsin-like proteases that cleave at the carboxy-terminal side of amino acid residues. hydrophobic. The proteases of the invention are serine endopeptidases (EC 3.4.21) with an acidic pH optimum (pH optimum < pH 7).

Protease activity can be measured using any assay, in which a substrate is employed, which includes peptide bonds relevant to the specificity of the protease in question. Also, the pH of the assay and the temperature of the assay must be adapted to the protease in question. Examples of test pH values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Examples of test temperatures are 15, 20, 25, 30, 35, 37, 40 , 45, 50, 55, 60, 65, 70, 80, 90 or 95°C. Examples of general protease substrates are casein, bovine serum albumin, and hemoglobin. In the classic method of Anson and Mirsky, denatured hemoglobin is used as a substrate and, after incubation of the assay with the protease in question, the amount of hemoglobin soluble in trichloroacetic acid is determined as a measure of protease activity (Anson, ML and Mirsky, AE, 1932, J. Gen. Physiol. 16:59 and Anson, ML, 1938, J. Gen. Physiol. 22:79).

For the purpose of the present invention, protease activity was determined using assays described in "Materials and Methods", such as the Kinetic Suc-AAPF-pNA assay, Protazyme AK assay, Kinetic Suc-AAPX-pNA assay and or -phthaldialdehyde (OPA). For the Protazyme AK assay, the insoluble Protazyme AK substrate (azurin cross-linked casein) releases a blue color when incubated with the protease and the color is determined as a measure of protease activity. For the Suc-AAPF-pNA assay, the colorless Suc-AAPF-pNA substrate releases yellow paranitroaniline when incubated with the protease and the yellow color is determined as a measure of protease activity.

Endo-Proteases/Exo-Proteases: Polypeptides that have protease activity, or proteases, are sometimes also called peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. Proteases can be of the exo type (exopeptidases) that hydrolyze peptides starting at either end, or of the endo type that act internally on polypeptide chains (endopeptidases).

S53 Protease: The term "S53" means a protease activity selected from:

(a) proteases belonging to the EC 3.4.21 group of enzymes; me

(b) proteases belonging to the EC 3.4.14 group of enzymes; me

(c) serine proteases of the S53 peptidase family comprising two different types of peptidases: tripeptidyl aminopeptidases (exo-type) and endo-peptidases; as described in 1993, Biochem. J. 290: 205-218 and in the MEROPS protease database, version 9.4 (January 31, 2011) (www.merops.ac.uk). The database is described in Rawlings, N.D., Barrett, A.J. and Bateman, A., 2010, "MEROPS: the peptidase database", Nucl. Acids Res. 38: D227-D233.

In determining whether a given protease is a Serine protease and an S53 family protease, reference is made to the above Manual and the principles stated therein. Such a determination can be carried out for all types of proteases, whether they are naturally occurring or wild-type proteases; or genetically modified or synthetic proteases.

Allelic Variant: The term "allelic variant" means any of two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic variation arises naturally through mutation and can result in polymorphism within populations. Gene mutations may be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a spliced, mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. The cDNA lacks intronic sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, beginning with a start codon such as ATG, GTG or TTG and ending with a stop codon such as TAA, TAG or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control Sequences: The term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide encoding a variant of the present invention. Each of the control sequences may be native ( ie, from the same gene) or heterologous ( ie, from a different gene) to the polynucleotide encoding the variant or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. The control sequences can be provided with linkers in order to introduce specific restriction sites that facilitate ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

Expression: The term "expression" includes any step involved in the production of a variant including, but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression Vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide that encodes a variant and is operably linked to control sequences that provide for its expression.

Fragment: The term "fragment" means a polypeptide that has one or more ( eg, several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has protease activity.

High stringency conditions: The term "high stringency conditions" means for probes at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sperm DNA of sheared and denatured salmon and 50% formamide following standard Southern blotting procedures for 12 to 24 hours. The support material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.

Host cell: The term "host cell" means any type of cell that is amenable to transformation, transfection, transduction or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a stem cell that is not identical to the stem cell due to mutations that occur during replication.

Improved Property: The term "improved property" means a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, increased specific activity, increased stability under storage conditions, increased thermostability, and increased expression levels in a recombinant expression host cell.

Isolated: The term "isolate" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, that has been removed, at least partially, from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by man-made relative to the substance found in nature or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated ( eg, multiple copies of a gene encoding the substance; use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). . An isolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term "low stringency conditions" means for probes at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sperm DNA of sheared and denatured salmon and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The support material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.

Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 199 to 564 of SEQ ID NO: 2. Amino acids 1 to 17 of SEQ ID NO: 2 are a signal peptide and amino acids 18 to 198 are a propeptide. The N-terminals of the mature S53 polypeptides used in accordance with the present invention were confirmed experimentally based on N-terminal EDMAN sequencing data and intact MS data. Mature polypeptides are also included as SEQ ID NO: 3 (Mature protease 3 S53 from Meripilus giganteus. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides ( i.e., with an amino acid C. different terminal and/or N-terminal), expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having protease activity. In one aspect, the mature polypeptide coding sequence is nucleotides 595 to 1692 of SEQ ID NO: 1. Nucleotides 1 to 51 of SEQ ID NO: 1 encode a signal peptide and nucleotides 52 to 594 encode a propeptide.

Medium stringency conditions: The term "medium stringency conditions" means for probes at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sperm DNA of sheared and denatured salmon and 35% formamide following standard Southern blotting procedures for 12 to 24 hours. The support material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.

Medium-high stringency conditions: The term "medium-high stringency conditions" means for probes at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/mL of sheared and denatured salmon sperm DNA and 35% formamide following standard Southern blot procedures for 12 to 24 hours. The support material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.

Mutant: The term "mutant" means a polynucleotide that encodes a variant.

Nucleic Acid Construct: The term "nucleic acid construct" means a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or modified to contain segments of nucleic acids. nucleic in a manner that would not otherwise exist in nature or that is synthetic, comprising one or more control sequences. In one embodiment, the one or more control sequences are heterologous (of different origin/species) to the coding sequence encoding the polypeptide of the invention.

Operably Linked: The term "operably linked" means a configuration in which a control sequence is placed in an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

Parental or Parental Protease: The term "parental" or the term "parental protease" means any polypeptide with protease activity in which an alteration is made to produce the enzyme variants of the present invention.

Sequence identity: The relationship between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and the substitution matrix EBLOSUM62 (EMBOSS version of BLOSUM62). The Needle output marked as "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residuals x 100)/(Alignment Length - Total Number of Holes in Alignment)

For purposes of the present invention, sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and the substitution matrix EDNAFULL (EMBOSS version of NCBI NUC4.4). The output of Needle marked "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Alignment Length - Total Number of Gaps in Alignment)

Subsequence: The term "subsequence" means a polynucleotide having one or more (eg, several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity. In one aspect, a subsequence contains at least 1098 nucleotides (eg, nucleotides 595 to 1692 of SEQ ID NO: 1).

Variant: The term "variant" means a polypeptide having protease activity that comprises a substitution at one or more ( eg, several) positions. A substitution means the replacement of the amino acid occupying one position with a different amino acid. Variants of the present invention have at least 20%, e.g. e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the protease activity of the mature polypeptide of SEQ ID NO: 2, described herein as SEQ ID NO: 3.

Very high stringency conditions: The term "very high stringency conditions" means for probes at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml DNA of sheared and denatured salmon sperm and 50% formamide following standard Southern blotting procedures for 12 to 24 hours. The support material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.

Very low stringency conditions: The term "very low stringency conditions" means for probes at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml DNA of sheared and denatured salmon sperm and 25% formamide following standard Southern blotting procedures for 12 to 24 hours. The support material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.

Wild-Type Protease: The term "wild-type" protease means a protease expressed by a naturally occurring microorganism, such as a bacteria, yeast, or filamentous fungus found in nature.

Variant Naming Conventions

For the purposes of the present invention, the mature polypeptide comprised in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another protease. The amino acid sequence of another protease is aligned with the mature polypeptide comprised in SEQ ID NO: 2 (described herein as SEQ ID NO: 3), and based on the alignment, the amino acid position number corresponding to any residue of amino acid in the mature polypeptide described as SEQ ID NO: 3 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the package EMBOSS (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and the substitution matrix EBLOSUM62 (EMBOSS version of BLOSUM62).

Identification of the corresponding amino acid residue in a protease other than the Meripilus giganteus S53 protease can be determined by an alignment of multiple polypeptide sequences using various computer programs including, but not limited to MUSCLE (multiple sequence comparison by sequence expectation). registry; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et a/., 2005 , Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et a/., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: _1899-1900), and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ ID NO: 2 such that traditional sequence-based comparison does not detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), they can be use other sequence comparison algorithms. Greater sensitivity in sequence-based searching can be achieved by using search programs that use probabilistic representations of families (profiles) of polypeptides to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologues (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even higher sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction , structural alignment profiles and solvation potentials) as input to a neural network that predicts the structural folding of a query sequence. . Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments, in turn, can be used to generate homology models for the polypeptide, and the accuracy of such models can be assessed using a variety of tools developed for that purpose.

For proteins of known structure, various tools and resources are available to retrieve and generate structural alignments. For example, the SCOP protein superfamilies have been structurally aligned, and those alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms such as distance alignment matrix (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11 : 739-747), and the implementation of these algorithms can be further used to query structure databases with a structure of interest in order to discover possible structural homologues ( e.g., Holm and Park, 2000, Bioinformatics 16 : 566-567).

In describing variants of the present invention, the nomenclature described below is adapted for ease of reference. The IUPAC accepted single-letter or three-letter amino acid abbreviation is used.

substitutions. For an amino acid substitution the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is designated "Thr226Ala" or "T226A". Multiple mutations are separated by addition marks ("+"), e.g. eg, "Gly205Arg Ser411 Phe" or "G205R S411F", which represent substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

deletions. For an amino acid deletion the following nomenclature is used: Original amino acid, position, *. Accordingly, the glycine deletion at position 195 with is designated "Gly195*" or "G195*". Multiple deletions are separated by addition marks (“+”), e.g. g., "Gly195*Ser411*" or "G195*S411*".

inserts. For an amino acid insertion the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Therefore the insertion of lysine after glycine at position 195 is designated "Gly195GlyLys" or "G195GK". An insertion of multiple amino acids is designated [Parent amino acid, position, parent amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine after glycine at position 195 is indicated as "Gly195GlyLysAla" or "G195GKA".

In such cases, the inserted amino acid residue(s) are numbered by adding lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the example above, the sequence would be:

multiple alterations. Variants comprising multiple alterations are separated by addition marks ("+"), e.g. eg, "Arg170Tyr Gly195Glu" or "R170Y G195E" representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

different alterations. In cases where different accidentals can be entered at one position, the different accidentals are separated by a comma, e.g. eg, "Arg170Tyr, Glu" represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala Arg170Gly,Ala" designates the following variants:

"Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to protease variants comprising a substitution at one or more ( eg, several) positions corresponding to specific positions in the mature polypeptide of SEQ ID NO: 2 (a parent protease), wherein the variant has protease activity. As explained herein, the specific position numbers may change in case the mature parent protease is different from SEQ ID NO: 3. The improved properties of the variants of the invention fall into the following categories, e.g. e.g., increased specific activity, increased stability, e.g. eg, increased storage stability or increased thermostability (measured as increase in half-life after heat stress (5 minutes at 60°C) as described in the examples) and increased expression levels in a recombinant expression host cell.

variants

The present disclosure provides protease variants that comprise a substitution at one or more positions corresponding to positions 1, 5, 9, 25, 29, 30, 32, 38, 39, 40, 41, 42, 45, 46, 49, 50, 53, 54, 55, 57, 59, 60, 61,

62, 64, 67, 68, 69, 74, 76, 77, 78, 79, 80, 81, 82, 87, 93, 94, 96, 99, 102, 103, 105, 109, 111, 112, 114, 115, 117, 123, 124, 128, 130, 136, 141, 142, 145, 146, 147, 150, 153, 154, 157, 159, 161, 167, 169, 175, 182, 185, 199, 200,

205, 207, 209, 210, 213, 216, 217, 225, 228, 231, 234, 237, 242, 244, 245, 246, 248, 258, 262, 266, 267, 272, 276, 278, 280, 284, 289, 293, 296, 299, 305, 308, 313, 314, 317, 318, 319, 321, 322, 324, 325, 326, 331, 333, 336, 338, 341,343, 345, 348, 347, 347 355, 358, 359, 361, 362, 363, and 364 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity. In one embodiment, the variant has increased thermostability measured as an enhancement factor, HIF, compared to the protease of SEQ ID NO: 3.

In one aspect of the disclosure, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% sequence identity. , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, of the amino acid sequence of the mature parental protease.

In another aspect, the variants have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at less than 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3.

In one aspect, the number of substitutions in the variants of the present invention is 1-20p . eg, 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

In one aspect, the disclosure relates to a protease variant comprising a substitution at one or more positions corresponding to positions 1, 5, 9, 25, 29, 30, 32, 38, 39, 40, 41, 42, 45, 46, 49, 50, 53, 54, 55, 57,

59, 60, 61,62, 64, 67, 68, 69, 74, 76, 77, 78, 79, 80, 81,82, 87, 93, 94, 96, 99, 102, 103, 105, 109, 111, 112, 114, 115,

117, 122, 123, 124, 128, 130, 136, 141, 142, 145, 146, 147, 150, 153, 154, 157, 159, 161, 167, 169, 175, 182, 185,

199, 200, 205, 207, 209, 210, 213, 216, 217, 225, 228, 231, 234, 237, 242, 244, 245, 246, 248, 258, 269, 271, 272, 276, 278, 280, 284, 289, 293, 296, 299, 305, 308, 313, 314, 317, 318, 319, 321, 322, 329, 330, 331,333, 336, 338, 341,343, 345, 347, 35,3548 358, 359, 361,362, 363, and 364 of the polypeptide of SEQ ID

NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, and wherein the variant has increased thermostability measured as enhancement factor, HIF, compared to the protease of SEQ ID NO: 3.

More specifically, the variant comprises a substitution at one or more positions selected from the group consisting of: A1T, S5I, S5K, S5L, S5Y, T9H, K25P, S29I, S29P, S29R, S30E, K32W, F38L, I39G, I39L, I39V, I39Y, D40F, D40G, D40H, D40N, D40P, Q41I, Q41V, F42C, F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, A46E, K49P, S50C, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54I, Q54L, Q54M, Q54V, F55H, F55L, K57P, K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C , E74H, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A, E81H, E81K, E81L, E81P, E81Y, A82F, A82L, A82M, A82P, N87S, T93L, T93S, V94S, L96W, G99C, T102L , T102V, T103i, T103K, T103V, I105Q, D109H, D109K, F111K, F111S, F111Y, Q112R, G114L, N115A, N115C, N115T, N115Y, E117D, E117G, E117H , E117N, E117Q, E117S, I122K, I122R, I123A, N124L, N124V, G128A, G128E, G128F, G128L, G128S, S130D, S130G, S130L, S130N, S130P, L136K, Q1421A, Q1421T, Q1421T, Q1421T, Q1421T N, N145V, T146A, T146I, T146L, T146N, I147S, K150F, K150L, K150R, N153A, N153L, N153M, N153P, N153R, N153Y, Q154A, Q154I, Q154L, Q154M, N157I, Q154N, Q154N, Q154N, Q154T Y159H, Q161E, T167F, I169F, I169S, D175I, Q175N, Q182C, H185P, F199L, F19M, M200S, A205S, Q207H, Q207L, V209F, S210T, T213L, T213R, A216R, F217i, F217Y, V225Y, F217Y, V225Y, F217Y, V225Y, F217Y, V225Y, F217Y, V225Y, F217Y, V225Y, F217Y, V225Y, F217Y, V225A I228L, I228T, I228W, Y2311, S234Q, S237F, S237Q, A242V, G244S, S245P, T246S, S248Y, F258P, S262P, V266K, V266R, D267A, D267H, D267L, D267N, Q269L, V271 R, S272I , S272K, S272L, S272R, S272T, S272V, T276Y, G278P, D280N, D280S, D280T, D280Y, C284A, C284G, F289Y, I293T, V296A, V296i, R299N, K305S, L308V, P313L, F314i, L308V, P313L, F314i, L308V, P313L, F314i, L308V, P313L, F314i , S317E, S317N, S317W, S318P, A319F, A319Q, K321S, A322D, L324Q, L324S, N325L, D326P, S329L, S329I, S329L, S329W, G330Q, S331W, S331Y , P333R, S336K, S336N, N338M, N338Q, N338R, P341S, K343A, K343H, K343S, K343T, K343V, G345S, D347I, P348Y, P355I, P355R, A358L, A3 58P, A358R, A358Y, K359L, K359S, L361M, T362L, T362N, A363G, A363L, and V364I, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein substitution at the one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 1.4, and further, wherein the variants have at least 90 %, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a more specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: S5K, S5L, S5Y, T9H, K25P, S29R, S30E, K32W, F38L, I39G, I39L, I39V, I39Y, D40F , D40G, D40H, D40N, D40P, Q41I, Q41V, F42C, F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, K49P, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54I, Q54L, Q54M , Q54V, F55H, F55L, K57P, K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A, E81H, E81K, E81L, E81P, E81Y, A82M, A82P, N87S, T93L, V94S, G99C, T102L, T102V, T103I, T103K, T103V, I105Q, D109H, D109M, D109Y , F111H, F111K, F111S, Q112R, G114L, N115C, N115K, E117D, E117G, E117H, E117N, E117Q, E117S, I122R, N124L, G128F, G128S, S130G, S130N, S130P, L136K, G141T , Q142A, Q142F, Q142L, Q142N, N145V, T146A, T146L, T146N, I147S, K150L, N153M, N153P, N153R, Q154I, Q154L, Q154N, Q154T, Q154V, N157L, Q161E, T167F F, D175N, Q182C, Q182T, Q182V, F199L, F199M, M200S, A205S, Q207H, Q207L, V209F, S210T, T213R, F217I, V225Y, I228L, I228T, I228W, S234Q, S237F, G245S, S248P, S2454S, S2454S, F258P S262P V266K V266R D267A D267H D267L V271 R S272K S272L S272R S272V S272Y , K305S, L308V, F314L, S317E, S317W, S318P, A319F, K321Q, K321S, A322D, L324Q, L324S, D326P, S329L, S329N, S329L, S329N, S329W, G330Q, S331W, S331Y, S336K , S336N, P341S, K343A, K343H, K343S, K343T, G345S, D347I, P348Y, P355I, A358L, A358P, A358R, A358Y, K359L, K359S, L361M, T362L, A363G, A363L, and V364I, where positions correspond to amino acid positions in the amino acid sequence listed in SEQ ID NO: 3; and wherein substitution at the one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 1.6, and further, wherein the variants have at least 90 %, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: S5K, S5L, S5Y, T9H, K25P, S30E, F38L, I39G, I39L, I39V, I39Y, D40F, D40G, D40H , D40N, D40P, Q41I, Q41V, F42K, F42Y, K45L, K45M, K45Y, A46C, K49P, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54L, Q54M, F55H, F55L, K57P, K57S, I59G , I59M, I59P, S60P, S61P, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C, E74R, D76L, D76R, Q77L, S78D, P79Y, E81A, E81K, E81L, E81P, E81Y, A82P, N87S, T93L, V94S, G99C, T102L, T102V, T103I, T103K, T103V, I105Q, D109H, D109K, D109M, D109Y, F111H, F111K, F111S, Q112R, G114L, N115R, E17N, E17N, E17N, E17N, E17N, E17N, E17N, I122R, N124L, G128S, S130G, S130N, S130p, L136K, Q142A, N145V, T146A, T146L, I147S, K150L, N153M, N153R, Q154i, Q154L, Q154N, Q154T, Q154V, N157L, Q161E, T167F I169F, D175N, Q182C, Q182V, F199L, F199M, M200S, Q207H, S210T, T213R, F217I, I228L, I228T, I228W, S237F, G244S, T246S, S248Y, F258P, S2 62P, V266K, V266R, D267A, D267H, D267N, V271R, S272K, S272L, S272R, S272V, S272Y, T276Y, G278P, D280N, D280S, D280T, C284G, F289Y, I293T, V31085L, L31085L, L31085L , S317W, S318p, A319F, A319Q, L324S, D326P, S329C, S329N, S329W, G330Q, S331W, S331Y, S336N, P341S, K343H, K343S, K343T, G345S, D347I, P355I, A358P, A358R, A358Y , K359L, K359S, T362L, A363L, and V364I, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein substitution at the one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 1.8, and further, in where the variants have at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% identity sequence with the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: S5K, S5Y, K25P, S30E, I39G, I39L, I39Y, D40F, D40G, D40H, D40N, D40P, Q411, Q41V , F42K, K45M, K45Y, A46C, S50I, S50L, S50M, S50Q, A53C, A53V, Q54M, F55H, F55L, K57P, K57S, I59M, I59P, S60P, S61P, T64I, Q68V, Q68Y, E74R, D76L, D76R ,S78D,P79Y,E81A,E81K,E81L,E81P,A82P,N87S,T93L,V94S,T102L,T102V,T103I,T103K,T103V,D109H,D109K,D109M,D109Y,F111H,F111K,F115GL,N1114GL,N1111S,G115GL , E117H, E117N, I122R, G128S, S130G, S130N, L136K, Q142L, Q142N, T146L, I147S, K150L, N153M, Q154T, Q154V, N157L, I169F, D175N, F199L, M200S , S210T, T213R, F217i, I228L, I228W, T246S, S262P, V266K, D267H, D267N, V271R, S272L, S272V, S272Y, T276Y, G278P, D280S, D280T, C284G, F289Y, I293T, V296A, S317E , S317W, S318P, A319F, L324S, D326P, S329C, S329I, S329W, G330Q, S331Y, S336N, P341S, K343T, D347I, P355I, A358 P, A358R, A358Y, K359L, T362L, and A363L, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein substitution at the one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 2.2, and further, wherein the variants have at least 90 %, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a more specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: S5K, I39G, I39L, I39Y, D40H, F42K, K45M, A46C, S50I, S50M, A53C, Q54M, F55H, F55L , K57P, K57S, I59M, I59P, T64I, Q68Y, E74R, D76L, S78D, P79Y, E81K, E81L, N87S, T93L, T102V, T103I, T103K, T103V, D109H, D109Y, F111H, F111R, E1, F1171S , E117H, I122R, L136K, Q142L, Q142N, T146L, I147S, N153R, Q154L, Q154N, Q154T, N157L, I169F, D175N, F199L, M200S, S210T, T213R, I228L, S27R, V27R, D2626S, S2626S, S272V, S272Y, T276Y, D280S, D280T, C284G, F289Y, I293T, V296A, S317E, S317W, A319F, L324S, S329I, S329W, S331Y, S336N, P341S, D347I, P355I, and T362L positions, where positions of amino acids in the amino acid sequence collected in SEQ ID NO: 3; and wherein substitution at the one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 3.2, and further, wherein the variants have at least 90 %, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: I39G, D40H, K45M, A46C, S50I, A53C, F55H, F55L, K57P, T64I, E74R, P79Y, T102V, N115R , E117H, I147S, N157L, D175N, F199L, S210T, I228L, D267N, D280T, C284G, V296A, S317E, A319F, S329I, P341S, D347I, and P355I, where positions correspond to amino acid positions in the amino acid sequence collected in SEQ ID NO: 3; and wherein substitution at the one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 6.4, and further, wherein the variants have at least 90 %, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitution selected from the group consisting of A1T, S5I, S5K, S5L, S5Y, T9H, K25P, S29I, S29P, S29R, S30E, K32W, F38L, I39G, I39L, I39V, I39Y, D40F, D40G, D40H, D40N, D40P, Q41I, Q41V, F42C, F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, A46E, K49P, S50C, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54I, Q54L, Q54M, Q54V, F55H, F55L, K57P, K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C, E74H, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A, E81H, E81K, E81L, E81P, E81Y, A82F, A82L, A82M, A82P, N87S, T93L, T93S, V94S, L96W, G99C, T102L, T102V, T103i, T103K, T103V, I105Q, D109H, D109K, D109m, D109Y, F111H, F111K, F111S, F111Y, N115C, N115K, N115P, E117D, E117G, E117H, E117N E117Q, E117S, I122K, I122R, I123A, N124L, N124V, G128A, G128S, S130D, S130G, S130L, S130N, S130P, L136K, G141T, Q142A, Q142F, Q142L, Q142N, N145V, T146A T146i, T146L, T146N, I147S, K150F, K150L, K150R, N153A, N153P, N153R, N153Y, Q154A, Q154M, Q154L, Q154M, Q154N, Q154L, Y159H, Q161E, T167F I169F, I169S, D175I, D175N, Q182C, Q182T, Q182V, H185P, M200S, A205S, Q207H, Q207L, V209F, S210T, T213L, T213R, A216R, F217i, F217Y, V225Y, I228L, I228T, I228W, I228W Y231i, S234Q, S237F, S237Q, A242i, A242L, S245P, T246S, S248Y, F258P, S262P, V266K, V266R, D267A, D267H, D267L, D267N, Q269L, V271 R, S272I, S272K, S272L, S272R , S272T, S272V, S272Y, T276L, T276Y, D280S, D280N, C284A, C284G, F289Y, I293T, V296A, V296i, R299N, K305S, L308V, P313L, F314i, S317W , S318p, A319F, A319Q, K321L, K321Q, L321S, L324S, N325L, D326P, S329C, S329H, S329T, S329V, S329W, G330Q, S331W, S331Y, P333R, S336K, S336N , N338M, N338Q, N338R, P341S, K343A, K343H, K343S, K343T, K343V, G345S, D347I, P348Y, P355I, P355R, A358L, A358P, A358R, A358Y , K359L, K359S, L361M, T362L, T362N, A363G, A363L, and V364I, where the positions correspond to amino acid positions in the amino acid sequence listed in SEQ ID NO: 3, and where the substitution in one or more positions provides a protease variant having an increase in thermostability measured as enhancement factor, HIF, of at least 1.4 compared to the parent protease.

The present disclosure provides protease variants that comprise a substitution at one or more positions corresponding to positions 20, 27, 29, 34, 40, 42, 45, 46, 50, 53, 54, 55, 58, 59, 60, 61.62, 66, 69, 76, 81.87,

93, 96, 98, 101, 102, 103, 109, 110, 115, 117, 122, 124, 125, 127, 128, 130, 136, 141, 142, 145, 146, 148, 149, 150,

277, 280, 285, 288, 291, 292, 293, 304, 305, 306, 307, 308, 310, 317, 318, 319, 321, 326, 327, 336, 338, 341, 344, 345, 348, 356, 363, y 364 del polipéptido de SEQ ID NO: 3, y la variante tiene actividad de proteasa. En una realización, las variantes tienen una actividad específica incrementada medida como factor de mejora, AIF, en comparación con la proteasa de153, 154, 157, 159, 161, 164, 167, 182, 183, 184, 185, 188, 199, 200, 207, 208, 209, 210, 211, 231, 237, 244, 249, 252, 253, 261, 266, 267, 272, 275, 276,

277, 280, 285, 288, 291, 292, 293, 304, 305, 306, 307, 308, 310, 317, 318, 319, 321, 326, 327, 336, 338, 341, 344, 345, 348, 356, 363, and 364 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity. In one embodiment, the variants have increased specific activity measured as enhancement factor, AIF, compared to the protease of

SEQ ID NO: 3.

In one aspect of the disclosure, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% sequence identity. , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, of the amino acid sequence of the mature parental protease.

In another aspect, the variants have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at less than 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3.

In one aspect, the number of substitutions in the variants of the present invention is 1-20p . eg, 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

In a specific aspect, the disclosure relates to a protease variant comprising a substitution at one or more positions corresponding to positions 20, 27, 29, 34, 40, 42, 45, 46, 50,

66, 69, 76, 81, 87, 93, 96, 98, 101, 102, 103, 109, 110, 115, 117, 122, 124, 125, 127,

344, 345, 348, 358, 359, 361,362, 363, y 364del polipéptido de SeQ ID NO: 3, y la variante tiene actividad de proteasa, y en donde la variante tiene al menos el 80%, al menos el 85%, al menos el 90%, al menos el 95%, al menos el 96%, al menos el208, 209, 210, 213, 216, 219, 228, 231, 237, 244, 249 , 252, 253, 261, 266, 267, 272, 2 288, 291, 292, 294, 296, 299, 303, 304, 305, 306, 307, 308, 310, 317, 318, 319, 321, 3

344, 345, 348, 358, 359, 361,362, 363, and 364 of the polypeptide of SeQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, and wherein the variant has increased specific activity measured as a factor of improvement, AIF, compared to the protease of SEQ ID NO: 3.

More specifically, the variant comprises a substitution at one or more positions selected from the group consisting of: G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, D40S, D40Y, F42Y,

K45L, K45Y, A46E, A46K, S50A, S50C, S50I, S50L, S50T, A53I, A53K, A53L, A53S, Q54L, F55R, D58M, I59M, I59P, S60C, S60E, S60P, S61D, S61P, S62C, S62N, S66F, S66L, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, E81S, N87F, T93S, L96F, L96S, L96T, T98L, P101F, P101L, P101M, P102C, T102i, T103V, D109P, D110W, N115A, N115D, E117F, E117G, E117L, E117W, E117 and I122L, N124D, N124I, N124K, N124L, N124M, N124T, N124V, N124T, N124V, F125K, F125L, F125M, L127C, L127P, G128N, G128R, G128S, S130F, S130I, S130N, L136C, G141A, G141C, G141F, G141L, G141M, G141Q, G141R, G141V, Q142A, Q142L, Q142A, Q142L Q142M, Q142S, Q142W, N145F, N145G, N145I, N145L, N145P, N145V, N145W, T146A, T146M, T146I, T146M, T146V, T146W, T146V, T146W, S148G, A149V, K150F N153D, N153F, N153G, N153R, N153Y, Q154C, Q154F, Q154G, Q154I, Q154K, Q154L, Q154M, Q154R, Q154T, Q154V, Q154W, Q154Y, N157A, N157E, N157FY, Y159M, Y159 59H, Y159S, Q161A, A164L, T167D, T167V, Q182F, S183A, S183M, A184D, A184F, A184H, A184I, A184R, A184S, A184T, A184V, A184W, A184Y, H185A, H185L, H185P, H185S, N188L, N188Q, F199L, M200F, M200L, Q207F, G208H, V209H, V209L, V209N, V209W, V209Y, S210E, S210L, S210R, S210T, S210Y, P211 F, T213F , T213H, T213i, T213L, T213N, T213R, T213S, T213W, T216Y, S219m, I228F, I228H, I228L I228M, I228Q, I228V, I228Y, Y231F, Y231S, S237F, S237I, S237K, S237L, G244L , G244N, G244P, G244R, G249C, N252F, N252K, N252S, R253C, V261A, V261G, V266C, V266R, D267S, S272I, S272K, S272L, S272M, S272Q, S272R, S272T, S272Y, Q275A, Q275F, Q275K, Q275L , Q275R, Q275V, T276F, T276R, T276V, I277L, I277V, D280F, D280S, D280T, A285L, A285S, T288C, T288I, T288V, S291V, V292C, I293M, I293Y, S294T, S294V, V296L , R299P, A303C, A303F, A303H, A303S, A303V, G304H, G304K, G304P, G304R, G304S, G304Y, K305C, K305F, K305H, K305I, K305L, K305M, K305S, K305T, K305Y, S306L, S306M, S306R, P307C, L308P, F310A, S317A, S317C, S317K, S317L, S317K, S317L, S317R, S317W, S317Y, S318N, S318R, A319F, S318N, S318R, A319F, A319W, K321R, K321S, D326P, V327C, V327T, S336R, N338H, N338S, P341 R, A344K, A344L, A344P, G345K, G345S, P348G, P348L, P348S, N356L, N356Y, A358F, A358L , A358P, K359C, K359L, K359S, K359W, L361E, L361M, L361P, L361R, L361S, L361T, L361V, T362A, T362C, T362H, T362I, T362K, T362L, T362M, T362RQ, T362RQ, T362P, T362RQ, T362Y, A363E, A363F, A363L, A363M, A363V, V364F, V364i, V364L, V364m, and V364S, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 1.37, and further, wherein the variants have at least the 90%, at least 95%

of identity, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100%, sequence identity with the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, F42Y, K45L , K45Y, A46E, A46K, S50A, S50L, A53L, Q54L, F55R, I59M, I59P, S60E, S60P, S61D, S61P, S62N, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P , D76V, D76Y, E81S, N87F, T93S, L96F, L96S, L96T, T98L, P101F, P101L, P101M, P101R, T102C, T102I, T103V, D109P, D110W, N115A, N115D, N115G, E117D, E117D, E1117D , E117L, E117N, E117P, E117S, E117W, E117Y, N124D, N124I, N124K, N124V, F125K, F125L, F125M, L127C, L127P, G128W, G128Y, S130D, S130F , S130I, S130N, L136C, G141A, G141C, G141M, G141Q, G141R, G141V, Q142A, Q142L, Q142M, Q142S, Q142W, N145L, N145G, N145R, N145S, N145V, N145W, N145S, N145V, N145R, N145S, N145V, N145R, N145S, N145V, N145W , T146A, T146C, T146H, T146I, T146M, T146N, T146Q, T146R, T146V, T146W, S148G, A149V, K150F, N153Y, Q154I, Q154K , Q154L, Q154M, Q154R, Q154W, N157F, Y159S, Q159H, T167D, Q182F, A184F, A184i, A184K, A184V, A184W, M200F, M200L, Q207F, G208H, G208Y, V209F , V209G, V209H, V209L, V209N, V209W, V209Y, S210E, S210L, S210R, S210T, T213F, T213i, T213L, T213N, T213R, T213S, T213W, A216F, S219m, I228F, I228H, I228L, I228M , I228Q, I228V, I228Y, Y231F, Y231S, S237F, S237I, S237K, S237L, G244N, G249C, N252F, V261A, V261G, V266C, V266R, D267S, S272I, S272L, S272M, S272Q, S272R, S272T, S272Y, Q275F , Q275K, Q275L, Q275R, Q275V, T276F, T276R, I276V, D280F, D280H, D280T, D280S, D280T, A285S, T288C, T288V, I293Y, S294T, S294V, A303F, G304H, G304K, G304p , G304R, G304S, K305C, K305F, K305H, K305i, K305S, K305T, S306L, S306R, P307C, L308P, L308T, F310M, S317C, S317G, S317H, S317i, S317K, S317L, S317R, S317W , S317Y, S318N, S318R, A319F, A319W, K321R, K321S, D326P, V327C, V327T, S336R, N338H, N338S, N338T, P3411, P341N, P341R, A34 4K, A344L, A344P, G345K, G345S, P348G, P348L, P348S, N356L, N356Y, A358L, K359S, K359W, L361P, L361R, T362C, T362H, T362I, T362K, T362M, T362FY, and T362FY, T362R, T362R where the positions correspond to amino acid positions in the amino acid sequence collected in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 2.0, and further, wherein the variants have at least the 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, K45Y, A46E , S50L, A53L, F55R, I59M, S60E, S60P, S61P, S62N, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, E81S, N87F, T93S, L96F, L96S, L96T , T98L, P101F, P101L, P101M, P101R, T102C, D102i, D110W, N115A, N115D, E117G, E117L, E117N, E117P, E117S, E117W, E117Y, N124I, N124K, N124L , N124M, N124V, F125K, F125L, F125M, L127C, L127P, G128W, G128S, S130F, S130i, S130N, L136C, G141A, G141C, G141F, G141L, G141V , Q142A, Q142L, Q142M, Q142S, Q142W, N145F, N145G, N145I, N145P, N145R, N145S, N145V, N145W, T146A, T146C, T146H, T146I, T146M, T146N, T146Q, T146R, T146V, T146W, S148G, A149V , N153Y, Q154I, Q154L, Q154R, N157F, Y159H, Y159S, T167D, Q182F, A184F, A184I, A184K, A184W, F199L, M200L, G208h, G208Y, V209F, V209G, V209H, V209L, V209N, V209Y, S210E, T213N, T213I, T213N, T213R, I228F, I228L, I228Q, I228V, I228Y, Y231F, Y231S, S237F, S237i, S237K, S237F, S237i, S237K S237L, G244N, G249C, V261A, V266R, D267S, S272I, S272L, S272T, S272Y, Q275K, Q275L, Q275R, Q275V, T276F, T276V, I277L, D280F, D280M, D285S, D280S, D280T, D285S T288V, I293Y, S294T, S294V, G304H, G304K, G304P, G304R, K305i, K305T, S306L, P307C, L308P, F310M, F310P, F310Y, S317A, S317C, S317G, S317H, S317i, S317K, S317i, S317K S317L, S317R, S317W, S317Y, S318N, S318R, A319F, A319W, D326P, V327C, V327T, S336R, N338H, N338S, N338T, P3411, P341N, P341 R, A344K, A344L, G345K, G345S, P348G , P348L, P348S, N356L, N356Y, A358L, K359S, L361P, L361R, T362C, T362H, T362I, T362Y, and V364M, where the positions correspond to amino acid positions in the amino acid sequence reported in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 2.2, and further, wherein the variants have at least the 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: G20L, G20R, S29A, S29C, S29N, S29P, A34N, A34R, A46E, S50L, F55R, I59M, S60E, S60P , S61P, S62N, S66M, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, E81S, N87F, L96F, L96S, L96T, T98L, P101F, P101L, P101M, P101R, T102C, T102I, T109VI, T1093 , N115A, N115D, N115G, E117D, E117F, E117G, E117L, E117N, E114P, N124K, N124V, F125L, F125M, L127P, G128N, G128S, G128W, G128Y, S130D, S130F, G141A, G141F , G141L, G141M, G141Q, G141R, Q142A, Q142S, Q142W, N145G, N145i, N145S, N145W, T146A, T146H, T146N, Y159H, Y159S, T167D, Q182F, F159L, G208H , G208Y, V209F, V209G, V209H, V209L, V209N, V209Y, S210L, T213i, I228L, I228Q, Y231S, S237F, S237I, S237K, S237L, G244N, V261A, V266R, D267S, S272L, S272T, S272Y , Q275K, Q275R, T276F, T276V, I277L, D280F, D280N, D280S, D280T, A285S, S294T, G304H, G304K , G304P, G304R, G304S, K305H, K305I, L308P, F310M, F310P, S317C, S317G, S317H, S317I, S317K, S317L, S317R, S317W, S317Y, A319F, K321S, D326P, V327C, S336R, N338H, P341I, P341N, P341R, G345K, G345S, P348G, P348S, N356Y, A358L, L361P, T362I, and V364M, where the positions correspond to amino acid positions in the amino acid sequence collected in SEQ ID NO : 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 2.6, and further, wherein the variants have at least the 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: S29N, A34N, A34R, I59M, S60P, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, N87F , L96F, L96S, T98L, P101L, P101M, T102C, T102I, D109P, N115A, E117D, E117F, E117G, E117L, E117N, E117P, E117W, E117Y, N124I, N124K, N124V, S1, G1208F, G1328F , G141F, G141L, G141M, G141Q, G141R, Q142M, Q142S, Q142W, N145i, N145S, T146H, T146N, T146W, Q154R, Y159S, T167D, Q182F, V209F, V209H, S237I, I228Q, Y231S, S237I , S237L, D267S, S272T, S272Y, T276F, T276V, D280F, D280S, G304H, G304p, K305H, F310M, F310P, S317C, S317G, S317I, S317K, S317L, S317R, S317W, S317Y, A319F , D326P, V327C, N338H, P341N, P341R, G345K, G345S, P348S, N356Y, and L361P, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 3.0, and further, wherein the variants have at least the 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: S29N, A34N, A34R, I59M, S60P, D76A, D76K, D76L, D76N, D76P, D76Y, N87F, L96F, D109P , E117F, E117G, E117L, E117N, E117P, E117W, E117Y, N124V, G141F, G141R, Q142L, Q142M, Q142S, Q142W, T146H, V209H, I228Q, T276F, D280T, A285S , S294T, F310M, F310P, S317C, S317I, S317K, S317L, S317R, S317W, D326P, N338H, P341N, P341R, G345K, G345S, P348S, N356Y, and L361P, where positions correspond to amino acid positions in the sequence of amino acids collected in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 3.8, and further, wherein the variants have at least the 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: A34N, A34R, D76A, D76K, D76L, D76N, D76Y, N87F, E117F, E117G, E117L, E117N, E117P, E117W where the positions correspond to amino acid positions in the amino acid sequence collected in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 4.5, and further, wherein the variants have at least the 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitution selected from the group consisting of: G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, D40S, D40Y, F42Y, K45L , K45Y, A46E, A46K, S50A, S50C, S50I, S50L, S50T, A53I, A53K, A53L, A53S, Q54L, F55R, D58M, I59M, I59P, S60C, S60E, S60P, S61D, S61P, S62C, S62N, S66F , S66L, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, E81S, N87F, T93S, L96F, L96S, L96T, T98L, P101F, P101L, P101M, P101R, T102C , T102i, T103V, D109P, D110W, N115A, N115D, E117D, E117F, E117G, E117S, E117W, E117Y, I122L, N124D, N124I, N124K, N124L, N124M, N124T, N124V , F125K, F125L, F125m, L127C, L127P, G128N, G128R, G128S, S130F, S130i, S130N, L136C, G141A, G141C, G141F, G141L, G141V, Q142A, Q142L , Q142M, Q142S, Q142W, N145F, N145G, N145I, N145L, N145P, N145R, N145S, N145V, N145W, T146A, T146C, T146H, T146I, T146M, T146N, T146Q, T146R, T146 N157AY N157m, N157y, Y159F, Y159G, Y159H, Y159S, T167D, T167V, Q182F, S183A, S183F, A184D, A184F, A184H, A184I, A184K, A184L, A184Q, A184R, A184S, A184T, A184R, A184S, A184T A184V, A184W, A184 and H185A, H185L, H185P, H185S, N188L, M200F, M200L, Q207F, G208H, G208Y, V209L, V209N, V209W, V209Y, S210E, S210L, S210R S210T, S210Y, P211 F, T213F, T213H, T213i, T213L, T213N, T213R, T213Y, A216F, A216L, S219m, I228F, I228H, I228V, I228Q, I228V, I228Y, Y231F, Y231S, S237F , S237I, S237K, S237L, G244L, G244N, G244P, G244R, G249C, N252F, N252K, N252S, R253C, V261A, V261G, V266C, V266R, D267S, S272I, S272K, S272L, S272M, S272Q, S272R, S272T, S272Y A2 85S, T288C, T288i, T288V, S291V, V292C, I293M, I293Y, S294T, S294V, V296L, R299P, A303C, A303V, G304H, G304K, G304p, G304R, G304S, G304Y, K305C, K305F, K305C, K305F K305H, K305I, K305L, K305M, K305S, K305T, K305Y, S306L, S306M, S306R, P307C, L308P, L308T, F310M, F310P, F310Y, S317A, S317H, S317G, S317L, S317R, S317W, S317Y, S318N, S318R, A319F, A319W, K321R, K321S, D326P, V327C, D326P, V327C V327T, S336R, N338h, N338S, N338T, P3411, P341N, P341 R, A344K, G345K, G345S, P348G, P348L, P348S, N356L, N356Y, A358F, A358L, A358P, K359 , L361E, L361M, L361P, L361R, L361S, L361T, L361V, T362A, T362C, T362H, T362I, T362K, T362L, T362M, T362P, T362Q, T362R, T362V, T362Y, A363M, A363E, A363F A363V, V364F, V364I, V364L, V364M, and V364S, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, and where substitution at one or more positions provides a variant of protease having an increase in specific activity measured as enhancement factor, AIF, of at least 1.37 compared to the parent protease.

In an additional particular aspect, the present disclosure relates to a protease variant comprising a substitution at one or more positions corresponding to positions 39, 57, 59, 66, 102, 111, 115, 267, 272, 275 or 280 of the polypeptide of SEQ ID NO: 3 and the variant has protease activity, and wherein the variant comprises a substitution selected from the group consisting of: I39L, K57P, I59M, S66T, T102V, F111H, N115K, D267N, S272Y, Q275K , D280S, and further wherein the variants have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, but less than 100% identity of the amino acid sequence of SEQ ID NO: 3.

In particular, the disclosure relates to a protease variant comprising a substitution at one or more positions corresponding to positions 39, 57, 59, 66, 102, 111, 115, 267, 272, 275 or 280 of the polypeptide of SEQ ID NO: 3 and the variant has protease activity, and wherein the variant comprises a substitution selected from the group consisting of: I39L, K57P, I59M, S66T, T102V, F111H, N115K, D267N, S272Y, Q275K, D280S, and, furthermore, wherein the variants have at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% of amino acid sequence identity of SEQ ID NO: 3, and wherein the variant has residual activity measured by increased thermostability after heat stress for 20 min at 56°C.

In another aspect, the disclosure relates to a protease variant comprising a substitution at one or more positions corresponding to positions 39, 57, 59, 66, 102, 111, 115, 267, 272, 275 or 280 of the polypeptide of SEQ ID NO: 3 and the variant has protease activity, and wherein the variant comprises a substitution selected from the group consisting of: I39L, K57P, I59M, S66T, T102V, F111H, N115K, D267N, S272Y, Q275K, D280S, and, furthermore, wherein the variants have at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% identity of the amino acid sequence of SEQ ID NO: 3, and wherein the variant has residual activity measured by increased thermostability after heat stress for 20 min at 56°C, wherein the residual activity is at least 50 %, particularly at least 60%, at least 70%, at least 80%.

In another aspect, the disclosure relates to a protease variant comprising a substitution at one or more positions corresponding to positions 39, 57, 59, 66, 102, 111, 115, 267, 272, 275 or 280 of the polypeptide of SEQ ID NO: 3 and the variant has protease activity, and wherein the variant comprises at least three substitutions selected from the group consisting of: I39L, K57P, I59M, S66T, T102V, F111H, N115K, D267N, S272Y, Q275K, D280S, and further wherein the variants have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, but less than 100% identity of the amino acid sequence of SEQ ID NO: 3, and wherein the variant has residual activity measured by increased thermostability after heat stress for 20 min at 56°C, wherein the residual activity is at least 50%, particularly at least 60%, at least 70%, at least 80%.

In another aspect, the disclosure relates to a protease variant comprising substitutions corresponding to I39L+S66T+T102V or D267N+S272Y Q275K+D280S of the polypeptide of SEQ ID NO: 3 and, furthermore, wherein the variants have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the sequence of amino acids of SEQ ID NO: 3, and wherein the variant has an increased thermostability measured residual activity after heat stress for 20 min at 56°C, wherein the residual activity is at least 50%, particularly at least 60%, at least 70%, at least 80%.

In another aspect, the disclosure relates to a protease variant comprising substitutions corresponding to:

I39L+ S66T T102V;

I39L+S66T+T102V+D267N S272Y+D280S;

I39L+K57P+I59M+S66T+T102V F111H+N115K+D267N+S272Y,

I39L+K57P+159M+S66T+T102V+F111H+N115K+D280S; or

D267N+S272Y+Q275K+D280S in the polypeptide of SEQ ID NO: 3 and, furthermore, wherein the variants have at least 90%, at least 95%, at least 96%, at least 97% identity , at least 98% or at least 99%, but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, and wherein the variant has residual activity measured by increased thermostability after thermal stress for 20 min at 56°C, where the residual activity is at least 50%, particularly at least 60%, at least 70%, at least 80%.

The present disclosure provides protease variants that comprise a substitution at one or more positions corresponding to positions 2, 17, 22, 41,41,42, 45, 46, 54, 55, 57, 59, 60, 61,62, 63, 64, 66, 69, 80, 82, 84, 87, 98, 99, 102, 103, 112, 114, 115, 129, 131, 134, 149, 159, 167, 169, 199, 200, 205, 207, 209, 211, 213, 217,242, 250, 266, 267, 269, 270, 271, 272, 274, 278, 279, 280, 284, 288, 291, 292, 294, 296, 301, 304, 314, 314, 314 329, 331, 333, 336, 362, and 363 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity. In one embodiment, the variants have an increased expression level measured as enhancement factor, EIF, compared to the protease of SEQ ID NO: 3.

In one aspect of the disclosure, the variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% sequence identity. , at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, of the amino acid sequence of the mature parent protease.

In another aspect, the variant is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%. , at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3.

In one aspect, the number of substitutions in the variants of the present invention is 1-20p . eg, 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

In a specific aspect, the disclosure relates to a protease variant comprising a substitution at one or more positions corresponding to positions 2, 17, 22, 41,41,42, 45, 46, 54, 55, 57, 59 , 60, 61,62, 63, 64, 66, 69, 80, 82, 84, 87, 98, 99, 102, 103, 112, 114, 115, 129, 131, 134, 149, 159, 167, 169 , 199, 200, 205, 207, 209, 211, 213, 217,242, 250, 266, 267, 269, 270, 271, 272, 274, 278, 279, 280, 284, 288, 291, 292, 2964 , 301, 307, 314, 329, 331, 333, 336, 362, and 363 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, and wherein the variant has a level of increased expression measured as enhancement factor, EIF, compared to the protease of SEQ ID NO: 3.

More specifically, the variant comprises a substitution at one or more positions selected from the group consisting of: I2L, A17P, P22V, Q41L, Q41T, Q41V, Q41Y, F42R, K45S, A46L, A46W, Q54R, Q54S, Q54V, Q54Y, F55L, F55N, F55T, F55Y, K57H, K57L, K57V, I59D, I59K, I59R, I59S, I59V, S60I, S60L, S60Q, S60R, S60Y, S61A, S61F, S62H, S62I, S62L, S62R, S62V, T63A, T63E, T63G, T63R, T63V, T64C, T64D, T64M, S66G, T69A, T69F, T69P, T69R, T69Y, S80G, S80L, S80N, S80T, A82H, I84G, N87P, T98C, G99V, T102A, T102H, T103F, T103i, T103V, Q112H, Q112M, Q112R, Q112S, G114A, G114C, G114L, G114P, E129V, E129Y, N131i, N131S, N131V, Q134A, Q134L, Q134R, A149D, Y159F, Y159L, T167A, Y159L, T167A T167L, T167S, T167W, I169S, F167W, M200L, A205S, Q207N, V209P, P211 F, P211S, T213, F217i, V266A, V266F, V266L, V266S, V267L, D267T, D267V , Q269C, Q269F, Q269I, Q269S, Q269T, Q269V, Q269X, I270A, I270C, I270L, I270S, V271F, V2711, V271K, V271M, V271S, V271Y, S272A, S272N, G274R, G278S, V279I, D280Y, C284A, T288A, T288C, T288G, S291V, V292S, V296R, V296T, I301T, P307T, F314L, S329D, S329V, S331A, S331G, S331T, P333V, S336G, S336L S336T, T362R, and A363T, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in expression level measured as enhancement factor, EIF, of at least 1.38, and further wherein the variants have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the sequence of amino acids of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: I2L, A17P, P22V, Q41L, Q41Y, F42R, Q54R, Q54S, Q54Y, F55N, F55T, K57L, K57V, I59D , I59S, I59V, S60L, S60Q, S60R, S60Y, S61F, S62L, S62V, T63A, T63E, T63R, T64C, T64D, T64M, Q112H, Q112M, Q112R, Q112S, G114A, G114C, G114L, G115LY, N115LY , E129S, E129V, E129Y, N131i, N131S, N134V, Q134R, A149D, T167A, T167S, T167W, F167S, V266F, V266L, V266S, V267L, D267F, D267L, D267T , D267V, Q269C, Q269F, Q269I, Q269S, Q269T, Q269V, Q269X, I270A, I270C, I270L, I270S, V271F, V2711, V271K, V271M, S272A, G274R, G278S, V298A, T288C, T288C, T288C, T288C, V296T, F314L, S329D, S329V, S331A, S331G, S331T, P333V, S336G, S336L, and S336T, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in expression level measured as enhancement factor, EIF, of at least 1.6, and further wherein the variants have at least the 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: I2L, A17P, P22V, Q41Y, F42R, K57L, I59S, T63A, T63R, Q112H, Q112M, Q112R, Q112S, G114A , G114L, G114P, E129L, E129S, E129Y, N131I, N131S, Q134R, T167W, K250R, V266A, V266L, V266T, D267F, D267L, D267T, D267V, Q269C, Q269F, Q269I, Q269T, Q269V, Q269T, Q269 , I270A, I270L, I270S, V271I, V271M, G274R, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein substitution at the one or more positions provides a protease variant having an increase in expression level measured as enhancement factor, EIF, of at least 1.75, and further wherein the variants have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the sequence of amino acids of SEQ ID NO: 3.

In a further specific embodiment, the variant comprises a substitution at one or more positions selected from the group consisting of: Q112M, Q112S, G114L, E129Y, N131I, Q134R, K250R, D267F, D267L, D267V, Q269C, Q269V, I270A, I270L , V271I, G274R, where the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3; and wherein the substitution at the one or more positions provides a protease variant having an increase in expression level measured as enhancement factor, EIF, of at least 1.9, and further wherein the variants have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the sequence of amino acids of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitution selected from the group consisting of: I2L, A17P, P22V, Q41L, Q41T, Q41V, Q41Y, F42R, K45S, A46L, A46W, Q54R, Q54S, Q54V, Q54Y, F55L , F55N, F55T, F55Y, K57H, K57L, K57V, I59D, I59K, I59R, I59S, I59V, S60I, S60L, S60Q, S60R, S60Y, S61A, S61F, S62H, S62I, S62L, S62R, S62V, T63A, T63E , T63G, T63R, T63V, T64C, T64D, T64M, S66G, T69A, T69F, T69P, T69R, T69Y, S80G, S80L, S80N, S80T, A82H, I84G, N87P, T98C, G99V, T102A, T102H, T103I, T103I , T103V, Q112H, Q112M, Q112R, Q112S, G114A, G114C, G114L, G114P, E129V, E129Y, N131i, N131S, N131V, Q134A, Q134L, Q154R, A149D, Y159F, Y159L, T167A, T167L , T167S, T167W, I169S, F199Y, M200L, A205S, Q207N, V209P, P211F, P211S, T213S, F217I, A242E, K250R, V266A, V266F, V266L, V266S, V266T, D267V, D267V, D267L, D267L, D267L, D267Y, D267F Q269C, Q269F, Q269I, Q269S, Q269T, Q269V, Q269X, I270A, I270C, I270L, I270S, V271F, V271I, V271K, V271M, V271S, V271Y, S272A, S272N, G274R, G278S, V 279i, D280Y and C284A, T288A, T288C, T288G, S291V, V292S, V296R, V296T, I301T, P307T, F314L, S329D, S329V, S331A, S331G, S331T, P333V, S336G, S336L, S336T, T362R, and A363T, wherein the positions correspond to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 3, and wherein substitution at one or more positions provides a protease variant having increased expression level measured as enhancement factor, EIF, of at least 1.38 compared to the parent protease.

Variants may further comprise one or more additional alterations at one or more (eg, several) other positions.

The amino acid changes may be of a minor nature, ie, conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a poly-histidine tract, an antigenic epitope, or a binding domain.

Therefore, even though protease variants according to the invention may only comprise a specific substitution that provides the improved property according to the invention, they may still have addition modifications leading to a variant protease having at least 80 %, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity, with the amino acid sequence of the mature parent protease, e.g. eg, the protease of SEQ ID NO: 3. Preferably, these additional modifications should not significantly change the improved properties of the variant protease.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine). ), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg , Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

Essential amino acids in a polypeptide can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In this latter technique, single alanine mutations are introduced into each of the molecule's residues, and the resulting mutant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acids of the enzyme. putative contact site. See, eg, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol.

224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.

In one aspect, the variant has improved (increased) specific activity compared to the parent enzyme.

In one aspect, the variant has improved (increased) storage stability compared to the parent enzyme.

In one aspect, the variant has improved (increased) thermostability compared to the parent enzyme.

In one aspect, the variant has enhanced (increased) expression levels in a recombinant expression host cell.

Parental proteases

The parent protease can be (a) a polypeptide having at least 80% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions to (i) the coding sequence of the mature polypeptide of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii); or (c) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the coding sequence of the mature polypeptide of SEQ ID NO: 1.

In one aspect, the parent has a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%. %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, who have protease activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g. g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, of the mature polypeptide of SEQ ID 2.

In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 3. In another aspect, the parent comprises or consists of amino acids 199 to 564 of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide that hybridizes under high stringency conditions, or very high stringency conditions with (i) the coding sequence of the mature polypeptide of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof, can be used to design nucleic acid probes to identify and clone DNA encoding a parent of strains from different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blot procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the full sequence, but should be at least 15, e.g. g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g. eg, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. Probes are typically labeled to detect the corresponding gene (eg, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.

A library of genomic DNA or cDNA prepared from such other strains can be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the libraries or separated DNA can be transferred and immobilized on nitrocellulose or other suitable support material. In order to identify a clone or DNA that hybridizes to SEQ ID NO: 1 or a subsequence thereof, the support material is used on a Southern blot.

For purposes of the present disclosure, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the coding sequence of the mature polypeptide of SEQ ID NO: 1; (iii) the cDNA sequence thereof; (iv) the full length complement thereof; or (v) a subsequence thereof; under high to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other means of detection known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe is nucleotides 595 to 1692 of SEQ ID NO: 1. In another aspect, the nucleic acid probe is a polynucleotide encoding the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or a fragment of it. In another aspect, the nucleic acid probe is SEQ ID NO: 1 or the cDNA sequence thereof.

In another aspect of the disclosure, the parent is encoded by a polynucleotide having a sequence identity with the coding sequence of the mature polypeptide of SEQ ID NO: 1 of at least 80%, at least 85%, at least 90%. %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

The parent may be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator(s). Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to the sites described in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environment. microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

In another aspect, the parent is an S53 protease from Meripilus giganteus, e.g. eg, the protease of SEQ ID NO: 2 or the mature polypeptide thereof, described herein as SEQ ID NO: 3.

Preparation of Variants

Variants can be prepared using any mutagenesis process known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more ( eg, several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be achieved in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation into the plasmid. polynucleotide. Usually, the restriction enzyme that digests the plasmid and the oligonucleotide is the same, allowing the sticky ends of the plasmid and the insert to ligate together. See, p. eg, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

Site-directed mutagenesis can also be achieved in vivo by methods known in the art. See, p. eg, US Patent Application Publication No. 2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773 776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newsletter. 43: 15-16.

Any site-directed mutagenesis process can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction involves the in vitro synthesis of a polynucleotide molecule designed to encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies, where oligonucleotides are synthesized and assembled on photoprogrammable microfluidic chips.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or rearrangement, followed by a relevant screening procedure, such as those described by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; ow O 95/22625. Other methods that can be used include error-prone PCR, phage display ( eg, Lowman et al., 1991, Biochemistry 30: 10832-10837; US Patent No. 5,223,409; WO 92/06204 ) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using methods standard in the art. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide.

Semi-synthetic gene construction is achieved by combining aspects of synthetic gene construction and/or site-directed mutagenesis and/or random mutagenesis and/or shuffling. The semi-synthetic construct is typified by a method using polynucleotide fragments that are synthesized, in combination with PCR techniques. Thus, defined regions of genes can be synthesized de novo, while other regions can be amplified using mutagenic site-specific primers, while still other regions can be subjected to error-prone or non-error-prone PCR amplification. The polynucleotide subsequences can then rearrange.

polynucleotides

The present invention also relates to polynucleotides encoding a variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operatively linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with those of the present invention. control sequences.

The polynucleotide can be manipulated in a variety of ways to provide expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of the polynucleotide. The promoter contains transcriptional control sequences that mediate expression of the variant. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.

Examples of promoters suitable for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters derived from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, acid-stable alpha-amylase. Aspergillus niger acids , Aspergillus niger or Aspergillus awamori glucoamylase ( glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase , Fusarium oxysporum trypsin-like protease (WO 96/00787), Amyloglucosidase from Fusarium venenatum (WO 00/56900), Daria from Fusarium venenatum (WO 00/56900), Quinn from Fusarium venenatum (WO 00/56900), Lipase from Rhizomucor miehei, Aspartic proteinase from Rhizomucor miehei, Beta-glucosidase from Trichoderma reesei, Trichoderma reesei celiobiohydrolase I, Trichoderma reesei celiobiohydrolase II , Tr endoglucanase I ichoderma reesei, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III , Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase , as well as the promoter NA2- tpi (a modified promoter of an Aspergillus neutral alpha-amylase gene, in which the untranslated leader has been replaced by an untranslated leader of an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated and hybrid promoters thereof.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the polynucleotide encoding the variant. Any terminator that is functional in the host cell can be used.

Preferred terminators for filamentous fungal host cells are derived from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase , Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

The control sequence can also be a stabilizing region of mRNA downstream of a promoter and upstream of the coding sequence of a gene that increases expression of the gene.

The control sequence may also be a leader, an untranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the variant. Any leader that is functional in the host cell can be used.

Preferred drivers for filamentous fungal host cells are derived from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase genes.

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' end of the variant coding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. . Any polyadenylation sequence that is functional in the host cell can be used.

Preferred polyadenylation sequences for filamentous fungal host cells are derived from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase , Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell's secretory pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked in translational reading frame with the segment of the coding sequence encoding the variant. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required, in cases where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell can be used.

Coding sequences for the signal peptide effective for filamentous fungal host cells are the sequences encoding the signal peptide obtained from the genes for neutral amylase , Aspergillus niger, nigerglucoamilasa of Aspergillus, TAKA amylase of Aspergillus oryzae, cellulase from Humicola insolens endoglucanase V Humicola insolens, lipase from Humicola lanuginosa aspartic proteinase from Rhizomucor miehei.

The control sequence may also be a propeptide coding sequence that encodes an N-terminal propeptide of a variant. The resulting polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the genes for Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase.

In cases where both signal peptide and propeptide sequences are present, the propeptide sequence is disposed at the N-terminus of the variant and the signal peptide sequence is disposed at the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulate expression of the variant relative to host cell growth. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi , the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter and the Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate and the metallothionein genes that are amplified by heavy metals. In these cases, the polynucleotide encoding the variant would be operatively linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences can be joined to produce a recombinant expression vector which may include one or more convenient restriction sites to allow insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located on the vector such that the coding sequence is operably linked to the appropriate control sequences for expression.

The recombinant expression vector can be any vector ( eg, a plasmid or virus) that can be conveniently subjected to recombinant DNA processes and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, eg, a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one which, when introduced into the host cell, integrates into the genome and replicates along with the chromosome(s) into which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome, or a transposon, can be used.

The vector preferably contains one or more selectable markers that allow easy selection of transformed, transfected, transduced cells or the like. A selectable marker is a gene whose product provides biocide or virus resistance, heavy metal resistance, prototrophy to auxotrophs, and the like.

Selectable markers for use in a filamentous fungal host cell include, but are not limited to amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine -5'-phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilate synthase), as well as equivalents thereof. Preferred use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae niaD, niiA, amdS and pyrG genes and a Streptomyces hygroscopicus bar gene.

The vector preferably contains one or more elements that allow integration of the vector into the genome of the host cell or autonomous replication of the vector in the cell independently of the genome.

For integration into the host cell genome, the vector may be dependent on the polynucleotide sequence encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration by homologous recombination into the host cell genome at a precise location(s) on the chromosome(s). To increase the probability of integration at a precise location, the integrating elements must contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. Integrating elements can be any sequence that is homologous to the target sequence in the host cell genome. Furthermore, the integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can be integrated into the host cell genome by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that mediates autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that allows a plasmid or vector to replicate in vivo.

Examples of useful origins of replication in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be achieved according to the methods described in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a variant. An increase in the number of copies of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene with the polynucleotide in cases where cells containing amplified copies of the selectable marker gene and thereby additional copies of the polynucleotide can be selected by culturing the cells in the presence of the appropriate selectable agent.

The processes used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those of skill in the art (see, eg, Sambrook et al., 1989, supra).

Host Cell

The present invention also relates to recombinant host cells comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences directing expression of a variant of the present invention. In a particular embodiment, the recombinant host cell comprises the polynucleotide encoding a trehalase polypeptide of the present invention, wherein said polynucleotide is heterologous (of different origin/species) to the host cell. A construct or vector comprising a polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described above. The term "host cell" encompasses any progeny of a stem cell that is not identical to the stem cell due to mutations that occur during replication. The choice of a host cell will largely depend on the gene encoding the variant and its source.

The host cell can be any cell useful in the recombinant production of a variant, e.g. eg, a prokaryote or a eukaryote.

The host cell may be a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth's Dictionary of The Fungi and Bisby, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. "Yeast" as used herein includes ascosporogenic yeast (Endomycetales), basidiosporogenic yeast, and yeast belonging to fungi imperfecta (Blastomycetes). Since the classification of yeasts may change in the future, for purposes of this invention yeast will be defined as described in Biology and Activities of Yeast (Skinner, Passmore and Davenport, eds., Soc. App. Bacteriol. Symposium Series N 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis cell. , Saccharomyces oviformis, or Yarrowia lipolytica.

The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all the filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is produced by elongation of the hyphae and carbon catabolism is obligatorily aerobic. In contrast, the vegetative growth of yeasts such as Saccharomyces cerevisiae occurs by budding from a unicellular thallus, and carbon catabolism may be fermentative.

The filamentous fungal cell may be a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia , Pyromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma.

For example, the filamentous fungal cell may be a cell of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis rivulosa pannocinta, , Ceriporiopsis subrufa, Ceriporiopsis subvermispora, inops Chrysosporium, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium um trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride

Fungal cells can be transformed by a process involving protoplast formation, protoplast transformation and cell wall regeneration in a manner known per se. Suitable processes for the transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, In Abelson, JN and Simon, MI, eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc. , New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Production Methods

The present invention also relates to methods of producing a variant, comprising: (a) culturing a recombinant host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant.

Host cells are cultured in a suitable nutrient medium for variant production using methods known in the art. For example, the cell may be cultured by shake flask culture or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under suitable conditions. that allow expressing and/or isolating the variant. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions ( eg, in American Type Culture Collection catalogs). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

The variant can be detected using methods known in the art that are specific for variants. These detection methods include, but are not limited to the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the variant.

The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium by conventional processes including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The variant can be purified by a variety of methods known in the art including, but not limited to chromatography ( eg, ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic processes ( p. eg, preparative isoelectric focusing), differential solubility. ( eg, ammonium sulfate precipitation), SDS-PAGE, or extraction (see, eg, Protein Purification, Janson and Ryden, eds., VCH Publishers, New York, 1989) to obtain substantially pure variants.

In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing the variant is used as a source of the variant.

Enzyme Compositions

The present invention also relates to compositions comprising a variant protease of the invention. Preferably, the compositions are enriched for such a protease. The term "enriched" indicates that the pullulanase activity of the composition has been increased, e.g. g., with an enrichment factor of at least 1.1.

The compositions may comprise the S53 variant protease as the main enzyme component, e.g. g., a one-component composition. Alternatively, the compositions may comprise multiple enzyme activities, such as the S53 variant protease and one or more ( eg, several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g. g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, alpha-amylase, beta-amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, glucoamylase , invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, protease, ribonuclease, transglutaminase, or xylanase. In one embodiment, the composition comprises a variant S53 protease of the invention and a carbohydrate source generating enzyme and optionally an alpha-amylase. In a particular embodiment, the composition comprises an S53 variant protease and a glucoamylase. Preferably, the enzyme activities comprised in the composition are selected from the variant S53 protease of the invention and one or more enzymes selected from the group consisting of glucoamylase, fungal alpha-amylase.

In one embodiment, the glucoamylase comprised in the composition is of fungal origin, preferably from a strain of Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii, or a strain of Trametes, preferably T. cingulata, or a strain of Pycnoporus, preferably P. sanguineus, or a strain of Gloeophyllum, such as G. serpiarium or G. trabeum, or a Nigrofomes strain.

In one embodiment, the glucoamylase is derived from Trametes, such as a Trametes cingulata strain, such as that shown in SEQ ID NO: 4 herein.

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 4 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 4 herein.

In one embodiment, the glucoamylase is derived from Talaromyces, such as a Talaromyces emersonii strain, such as that shown in SEQ ID NO: 5 herein.

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 5 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 5 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as that shown as SEQ ID NO: 4 in WO 2011/066576.

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 6 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 6 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strain of Gloeophyllum, as described in WO 2011/068803 (SEQ ID NO: 2 , 4, 6, 8, 10, 12, 14 or 16). In a preferred embodiment, the glucoamylase is Gloeophyllum sepiarium shown in SEQ ID NO: 2 in WO 2011/068803 or SEQ ID NO: 7 in this specification.

In one embodiment, the glucoamylase is derived from Gloeophyllum serpiarium, such as that shown in SEQ ID NO: 7 herein. In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 7 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 7 herein.

In another embodiment, the glucoamylase is derived from Gloeophyllum trabeum such as that shown in SEQ ID NO: 8 herein. In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 8 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g. e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 8 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. described in WO 2012/064351.

In one embodiment, glucoamylases can be added to the saccharification and/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from DuPont).

In addition to a glucoamylase, the composition may further comprise an alpha-amylase. Particularly, alpha-amylase is an acidic fungal alpha-amylase. A fungal acid-stable alpha-amylase is an alpha-amylase having activity in the pH range of 3.0 to 7.0 and preferably in the pH range of 3.5 to 6.5, including activity at a pH of approximately 4.0, 4.5, 5.0, 5.5 and 6.0.

Preferably, the fungal acid alpha-amylase is derived from the genus Aspergillus, especially a strain of A. terreus, A. niger, A. oryzae, A. awamori, or Aspergillus kawachii, or from the genus Rhizomucor, preferably a strain of Rhizomucorpusillus, or the genus Meripilus, preferably a strain of Meripilus giganteus.

In a preferred embodiment, the alpha-amylase is derived from a strain of the genus Rhizomucor, preferably a strain of Rhizomucor pusillus, such as that shown in SEQ ID NO: 3 in WO 2013/006756, such as a hybrid Rhizomucor pusillus alpha-amylase enzyme having an Aspergillus niger linker and starch-binding domain, such as that shown in SEQ ID NO: 9 herein, or a variant thereof.

In one embodiment, the alpha-amylase is selected from the group consisting of:

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 9 herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 9 herein.

In a preferred embodiment, the alpha-amylase is a variant of the alpha-amylase shown in SEQ ID NO: 9 having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H Y141W; G20S Y141W; A76G Y141W; G128D Y141W; G128D D143N; P219C Y141W; N142D D143N; Y141W K192R; Y141W D143N; Y141W N383R; Y141W P219C A265C; Y141W N142D D143N; Y141W K192R V410A; G128D Y141W D143N; Y141W D143N P219C; Y141W D143N K192R; G128D D143N K192R; Y141W D143N K192R P219C; G128D Y141W D143N K192R; or G128D Y141W D143N K192R P219C (using SEQ ID NO: 9 for numbering).

In one embodiment, the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably described as SEQ ID NO: 9 herein, preferably with one or more of the following substitutions: G128D, D143N, preferably G128D D143N (using SEQ ID NO: 9 for numbering), and wherein the alpha-amylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85 %, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID NO: 9 herein.

In a preferred embodiment, the ratio of glucoamylase to alpha-amylase present and/or added during saccharification and/or fermentation may preferably be in the range of 500:1 to 1:1, such as 250:1 to 1:1. such as 100:1 to 1:1, such as 100:2 to 100:50, such as 100:3 to 100:70.

The compositions can be prepared according to methods known in the art and can be in the form of a liquid or dry composition. For example, the composition may be in the form of granules or microgranules. The variant can be stabilized according to methods known in the art.

The compositions can be prepared according to methods known in the art and can be in the form of a liquid or dry composition. The compositions can be stabilized according to methods known in the art.

The enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cell debris, a semi-purified enzyme composition or purified, or a host cell, as the source of the enzymes.

The enzyme composition may be a dry powder or granule, a non-dusting granule, a liquid, a stabilized liquid or a stabilized protected enzyme. Liquid enzyme compositions can, for example, be stabilized by adding stabilizers such as a sugar, sugar alcohol or other polyol and/or lactic acid or other organic acid according to established procedures.

Use of the variant proteases of the invention

Starch Processing

Native starch consists of microscopic granules, which are insoluble in water at room temperature. When the aqueous suspension of starch is heated, the granules swell and eventually burst, dispersing the starch molecules in solution. At temperatures up to about 50°C to 75°C, the swelling may be reversible. However, at higher temperatures an irreversible swelling called "gelatinization" begins. During this "gelatinization" process there is a drastic increase in viscosity. The granular starch to be processed may be a highly refined grade of starch, preferably at least 90%, at least 95%, at least 97%, or at least 99.5% pure or may be cruder starch-containing materials comprising ( p .g., ground) whole grains, including non-starchy fractions such as germ residues and fibers. Raw material such as whole grains can be reduced in particle size, e.g. eg., by milling, in order to open up the structure and allow further processing. In dry milling, whole grains are ground and used. Wet milling gives a good separation of germ and flour (starch and protein granules) and is often applied where starch hydrolyzate is used in the production of e.g. eg syrups. Both dry and wet milling are well known in the art of starch processing and can be used in a process of the invention. Methods for reducing the particle size of starch-containing material are well known to those skilled in the art.

As the solids level is 30-40% in a typical industrial process, the starch must be diluted or "liquefied" so that it can be processed properly. This viscosity reduction is achieved primarily by enzymatic degradation in current commercial practice.

Liquefaction is carried out in the presence of an alpha-amylase, preferably a bacterial alpha-amylase and/or acid fungal alpha-amylase. In one embodiment, a phytase is also present during liquefaction. In one embodiment, viscosity-reducing enzymes such as xylanase and/or beta-glucanase are also present during liquefaction.

During liquefaction, long-chain starch is broken down into shorter branched and linear units (maltodextrins) by alpha-amylase. Liquefaction can be carried out as a three stage hot slurry process. The suspension is heated to 60-95°C ( eg, 70-90°C, such as 77-86°C, 80-85°C, 83-85°C) and an alpha-amylase is added to initiate liquefaction (dilution).

In one embodiment, the slurry can be jet-cooked to between 95-140°C, eg. eg, 105-125°C, for about 1-15 minutes, eg, about 3-10 minutes, especially about 5 minutes. The suspension is then cooled to 60-95°C and more alpha-amylase is added to obtain the final hydrolysis (secondary liquefaction). The jet cooking process is carried out at pH 4.5-6.5, typically between pH 5 and 6. The alpha-amylase can be added as a single dose, e.g. e.g. before jet cooking.

The liquefaction process is carried out at 70-95°C, such as 80-90°C, such as about 85°C, for about 10 minutes to 5 hours, typically 1-2 hours. The pH is between 4 and 7, such as between 4.5 and 5.5. In order to ensure optimal enzyme stability under these conditions, calcium may optionally be added (to provide 1-60 ppm free calcium ions, such as about 40 ppm free calcium ions). After such a treatment, the liquefied starch will typically have a "dextrose equivalent" (DE) of 10-15.

Generally, liquefaction and liquefaction conditions are well known in the art.

Alpha-amylases for use in liquefaction are preferably bacterial acid-stable alpha-amylases. Particularly, the alpha-amylase is from an Exiguobacterium sp. or a Bacillus sp., such as, eg, Bacillus stearothermophilus or Bacillus licheniformis.

In one embodiment, the alpha-amylase is from the genus Bacillus, such as a strain of Bacillus stearothermophilus, in particular a variant of a Bacillus stearothermophilus alpha-amylase, such as that shown in SEQ ID NO: 3 in document WO 99/019467 or SEQ ID NO: 10 in this specification.

In one embodiment, the Bacillus stearothermophilus alpha-amylase has a double deletion of two amino acids in the region from position 179 to 182, more particularly a double deletion at positions 1181 G182, R179 G180, G180 1181, R179 1181 or G180 G182. , preferably 1181 G182, and optionally a N193F substitution (using SEQ ID NO: 10 for numbering).

In one embodiment, the Bacillus stearothermophilus alpha-amylase has a substitution at position S242, preferably an S242Q substitution.

In one embodiment, the Bacillus stearothermophilus alpha-amylase has a substitution at position E188, preferably an E188P substitution.

In one embodiment, the alpha-amylase is selected from the group of Bacillus stearothermophilus alpha-amylase variants with the following mutations:

• I181*+G182*+N193F+E129V+K177L+R179E;

• I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L Q254S;

• I181*+G182*+N193F V59A Q89R+ E129V+ K177L+ R179E+ Q254S+ M284V; Y

• I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 10 for numbering).

In one embodiment, the alpha-amylase variant has at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%. %, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99% , but less than 100% identity with the polypeptide of SEQ ID NO: 10.

It should be understood that when referring to Bacillus stearothermophilus alpha-amylase and variants thereof, they are normally produced in a truncated form. In particular, the truncation may be such that the Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 10 herein, or variants thereof, are truncated at the C-terminal preferably to be about 490 amino acids, such as 482-493 amino acids. Preferably, the Bacillus stearothermophilus variant alpha-amylase is truncated, preferably after position 484 of SEQ ID NO: 10, particularly after position 485, particularly after position 486, particularly after position 487, particularly after position 488, particularly after position 489, particularly after position 490, particularly after position 491, particularly after position 492, more particularly after position 493.

Saccharification can be carried out using conditions well known in the art with a carbohydrate source generating enzyme, in particular a glucoamylase or a beta-amylase and optionally a debranching enzyme, such as an isoamylase or a pullulanase. For example, a complete saccharification step can take from about 24 to about 72 hours. However, it is common to do pre-saccharification for typically 40-90 minutes at a temperature between 30-65°C, typically around 60°C, followed by full saccharification during fermentation in a simultaneous saccharification and fermentation (SSF) process. . Saccharification is typically carried out at a temperature in the range of 20-75°C, eg. eg, 25-65°C and 40-70°C, typically about 60°C, and at a pH between about 4 and 5, usually about pH 4.5.

The saccharification and fermentation steps can be carried out sequentially or simultaneously. In one embodiment, saccharification and fermentation are performed simultaneously (referred to as "SSF"). However, it is common to perform a pre-saccharification step for approximately 30 minutes to 2 hours ( eg, 30 to 90 minutes) at a temperature of 30 to 65°C, typically around 60°C, followed by a saccharification. complete during fermentation, known as simultaneous saccharification and fermentation (SSF). The pH is usually between 4.2 and 4.8, e.g. g., pH 4.5. In a simultaneous saccharification and fermentation (SSF) process, there is no holding phase for the saccharification, rather the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced during liquefaction are converted to dextrose by the addition of a glucoamylase and debranching enzyme, such as isoamylase (US Patent No. 4,335,208) or pullulanase. The temperature is reduced to 60°C before the addition of glucoamylase and debranching enzyme. The saccharification process continues for 24-72 hours. Before the addition of the saccharifying enzymes, the pH is lowered below 4.5, while maintaining a high temperature (above 95°C), to inactivate the liquefying alpha-amylase. This procedure reduces the formation of short oligosaccharides called "panose precursors", which cannot be adequately hydrolyzed by the debranching enzyme. Typically, about 0.2-0.5% of the saccharification product is the branched panose trisaccharide (Glc pa1-6Glc pa1-4Glc), which cannot be degraded by a pullulanase. If liquefaction active amylase remains present during saccharification ( ie without denaturation), the amount of panose can be as high as 1-2%, which is highly undesirable as it significantly reduces the saccharification yield.

Other fermentation products can be fermented under conditions and temperatures well known to those skilled in the art, suitable for the fermenting organism in question.

The fermentation product can be recovered by methods well known in the art, e.g. g., by distillation. Examples of carbohydrate source generating enzymes are described in the "Enzymes" section below.

In a particular aspect, the method of the invention further comprises, before the conversion of a starch-containing material into sugars/dextrins, the steps of:

(x) reducing the particle size of the starch-containing material; and

(y) forming a suspension comprising the starch-containing material and water.

In one embodiment, the starch-containing material is milled to reduce the particle size. In one embodiment, the particle size is reduced to between 0.05-3.0mm, preferably 0.1-0.5mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material passes through a sieve with a mesh size of 0.05-3.0 mm, preferably a mesh size of 0.1-0.5 mm .

The aqueous suspension may contain 10-55 wt% dry solids (DS), preferably 25-45 wt% dry solids (DS), more preferably 30-40 wt% dry solids (DS) of material that contains starch. Conventional starch conversion processes, such as liquefaction and saccharification processes, are described, e.g. eg, in US Patent No. 3,912,590, EP 252730 and EP 063909.

In one embodiment, the conversion process that degrades starch into lower molecular weight carbohydrate components, such as sugars or fat substitutes, includes a debranching step.

In the case of converting starch to a sugar, the starch is depolymerized. Such a depolymerization process consists, e.g. in a pretreatment step and two or three consecutive process steps, ie a liquefaction process, a saccharification process and, depending on the desired final product, an optional isomerization process.

When the final desired sugar product is e.g. For example, a high fructose syrup, dextrose syrup can be converted to fructose. After the saccharification process, the pH is increased to a value in the range of 6-8, eg. g., pH 7.5, and calcium is removed by ion exchange. The dextrose syrup is then converted to high fructose syrup using e.g. eg, an immobilized glucose isomerase.

Production of Fermentation Products

Fermentable sugars ( eg, dextrins, monosaccharides, particularly glucose) are produced from enzymatic saccharification. These fermentable sugars can be further purified and/or converted to useful sugar products. In addition, sugars can be used as a fermentation feedstock in a microbial fermentation process to produce end products such as alcohol ( eg, ethanol and butanol), organic acids ( eg, succinic acid , 3-HP, and lactic acid), sugar alcohols ( eg, glycerol), ascorbic acid intermediates ( eg, gluconate, 2-keto-D-gluconate, 2,5-diketo-D- gluconate and 2-keto-L-gluconic acid), amino acids ( eg, lysine), proteins ( eg, antibodies and fragments thereof). In one embodiment, the fermentable sugars obtained during the liquefaction process steps are used to produce alcohol and particularly ethanol. An SSF process is commonly used in ethanol production, in which saccharifying enzymes and fermenting organisms ( eg yeast) are added together and then carried out at a temperature of 30-40°C.

The organism used in the fermentation will depend on the desired end product. Typically, if ethanol is the desired end product, yeast will be used as the fermenting organism. In some preferred embodiments, the ethanol-producing microorganism is a yeast and specifically Saccharomyces such as S. cerevisiae strains (US Patent No. 4,316,956). A variety of S. cerevisiae are commercially available and these include, but are not limited to FALI (Fleischmann's yeast), SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre), and Angel alcohol yeast (Angel Yeast Company). , China) . The amount of initial yeast used in the methods is an amount effective to produce a commercially significant amount of ethanol in a suitable amount of time ( e.g., to produce at least 10% ethanol from a substrate that is between 25-40 % DS in less than 72 hours). The yeast cells are generally supplied in amounts of from about 104 to about 1012, and preferably from about 107 to about 1010 viable yeast counts per mL of fermentation broth. After the yeast is added to the mash, this is typically fermented for about 24-96 hours, e.g. eg, 35-60 hours. The temperature is between about 26-34°C, typically about 32°C, and the pH is pH 3-6, eg. eg, around pH 4-5.

Fermentation may include, in addition to fermenting microorganisms ( eg, yeast), additional nutrients and enzymes, including phytases. The use of yeast in fermentation is well known in the art.

In further embodiments, the use of appropriate fermenting microorganisms, as known in the art, can result in a fermentation end product including, e.g. g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gluconic acid, succinic acid, lactic acid, amino acids and derivatives of the same. More specifically, when lactic acid is the desired end product, a Lactobacillus sp. ( L. casei) when the desired end products are glycerol or 1,3-propanediol, E. coli can be used; and when 2-keto-D-gluconate, 2,5-diketo-D-gluconate and 2-keto-L-gluconic acid are the desired end products, Pantoea citrea can be used as the fermenting microorganism. The above listed list is exemplary only and one skilled in the art will be aware of a number of fermenting microorganisms that can be used to obtain a desired end product.

Procedures for producing fermentation products from ungelatinized starch-containing material

The invention relates to processes for producing fermentation products from starch-containing material without gelatinizing ( ie without cooking) the starch-containing material (often referred to as a "crude starch hydrolysis" process). . The fermentation product, such as ethanol, can be produced without liquefying the aqueous suspension containing the starch-containing material and water. In one embodiment, a method of the invention includes saccharifying ( eg, ground) starch-containing material, e.g. g., granular starch, below the initial gelatinization temperature, preferably in the presence of alpha-amylase and/or carbohydrate source-generating enzymes to produce sugars that can be fermented into the fermentation product by a fermentation organism. proper fermentation. In this embodiment, the desired fermentation product, e.g. eg, ethanol, is produced from ungelatinized ( ie, uncooked) cereal grains, preferably ground, such as corn.

Accordingly, in one aspect, the invention relates to processes for producing a fermentation product from starch-containing material, comprising simultaneously saccharifying and fermenting starch-containing material using an enzyme-generating carbohydrate source and an organism. fermenter at a temperature below the initial gelatinization temperature of said starch-containing material in the presence of a variant protease of the invention. Saccharification and fermentation can also be separate. Thus, in another aspect, the invention relates to processes for preparing fermentation products, comprising the following steps:

(i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature using a carbohydrate source-generating enzyme, e.g. eg, a glucoamylase; Y

(ii) fermenting using a fermenting organism;

wherein step (i) is carried out using at least one glucoamylase and one variant protease of the invention.

In one embodiment, the fermenting organism expresses the variant protease of the invention.

In one embodiment, an alpha amylase is also added in step (i). Steps (i) and (ii) can be performed simultaneously.

The fermentation product, e.g. eg ethanol, can optionally be recovered after fermentation, e.g. g., by distillation. Typically, amylase(s), such as glucoamylase(s) and/or other carbohydrate source-generating enzymes, and/or alpha-amylase(s), are present during fermentation. Examples of glucoamylases and other carbohydrate source-generating enzymes include glucoamylases that hydrolyze crude starch. Examples of alpha-amylase(s) include acid alpha-amylases, such as fungal acid alpha-amylases. Examples of fermentative organisms include yeasts, e.g. eg, a strain of Saccharomyces cerevisiae. The term "initial gelatinization temperature" means the lowest temperature at which starch gelatinization begins. In general, starch heated in water begins to gelatinize between about 50°C and 75°C; the exact gelatinization temperature depends on the specific starch and can be readily determined by those skilled in the art. Therefore, the initial gelatinization temperature may vary according to the species. plant, the particular variety of the plant species, as well as the growing conditions. In the context of this invention, the initial gelatinization temperature of a given starch-containing material can be determined as the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starch/Starke 44(12): 461-466. Before starting the process, a suspension of starch-containing material can be prepared, such as granulated starch, having 10-55% w/w dry solids (DS), preferably 25-45% w/w dry solids, more preferably 30-40% w/w dry solids of starch-containing material. The slurry may include water and/or process waters, such as stillage (backflow), scrubbing water, evaporator condensate or distillate, distillation side draw water, or process water from other fermentation products plants. Because the process of the invention is carried out below the initial gelatinization temperature and therefore no significant increase in viscosity occurs, high levels of vinasse can be used if desired. In one embodiment, the aqueous suspension contains from about 1 to about 70 vol.%, preferably 15-60 vol.%, especially from about 30 to 50 vol. of water and/or process waters, such as stillage (backflow), wash water, evaporator condensate or distillate, distillation side draw water, or process water from other fermentation product plants, or combinations thereof , or the like. The starch-containing material can be prepared by reducing the particle size, preferably by wet or dry milling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After having undergone a procedure of the invention, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids in the starch-containing material are converted to a soluble starch hydrolyzate. A process in this aspect of the invention is carried out at a temperature below the initial gelatinization temperature, which means that the temperature is typically in the range between 30-75°C, preferably between 45-60°C. In a preferred embodiment, the process is carried out at a temperature of from 25°C to 40°C, such as from 28°C to 35°C, such as from 30°C to 34°C, preferably around 32°C. c. In one embodiment, the process is carried out such that the sugar level, such as the glucose level, is maintained at a low level, such as below 6% w/w, such as below about 3% w/w, such as below about 2% w/w, such as below about 1% w/w, such as below about 0.5% w/w, or below 0 0.25% w/w, such as below about 0.1% w/w. Such low sugar levels can be achieved simply by employing adjusted amounts of enzyme and fermenting organism. A person skilled in the art can easily determine what dose/amounts of enzyme and fermenting organism to use. The amounts of enzyme and fermenting organism employed can also be selected to maintain low maltose concentrations in the fermentation broth. For example, the maltose level can be kept below about 0.5% w/w, such as below about 0.2% w/w. The process of the invention can be carried out at a pH of from about 3 to 7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In one embodiment, the fermentation is ongoing for 6 to 120 hours, in particular 24 to 96 hours.

Procedures for producing fermentation products from gelatinized starch-containing material

In this aspect, the invention relates to processes for producing fermentation products, especially ethanol, from starch-containing material, which process includes a liquefaction step and saccharification and fermentation steps performed sequentially or simultaneously. Consequently, the invention relates to a process for preparing a fermentation product from starch-containing material, comprising the steps of:

(a) liquifying starch-containing material in the presence of an alpha-amylase;

(b) saccharifying the liquefied material obtained in step (a) using an enzyme that generates a source of carbohydrates;

(c) fermenting using a fermenting organism;

wherein a variant protease of the invention is present during step b) or c).

In one embodiment, the fermenting organism expresses the variant protease of the invention.

The fermentation product, such as especially ethanol, can optionally be recovered after the fermentation, e.g. g., by distillation. The fermenting organism is preferably yeast, preferably a strain of Saccharomyces cerevisiae. In a particular aspect, the process of the invention further comprises, before step (a), the steps of:

x) reducing the particle size of the starch-containing material, preferably by grinding (eg using a hammer mill);

y) forming a suspension comprising the starch-containing material and water.

In one embodiment, the particle size is smaller than a #7 sieve, eg. eg, a No. 6 sieve. A No. 7 sieve is commonly used in conventional prior art processes. The slurry may contain from 10 to 55, mp. eg, 25-45 and 30-40% w/w dry solids (DS) of starch-containing material. The suspension is heated above the gelatinization temperature and an alpha-amylase variant can be added to initiate liquefaction (dilution). In one embodiment, the suspension may be jet-cooked to further gelatinize the suspension prior to subjecting it to alpha-amylase in step (a). The liquefaction can be carried out, in one embodiment, as a three-stage hot slurry process. The suspension is heated to 60-95°C, preferably 70-90°C, such as preferably 80-85°C at pH 4-6, preferably 4.5-5.5, and alpha-amylase variant, optionally together with a pullulanase. and/or protease, preferably metalloprotease, are added to initiate liquefaction (dilution). In one embodiment, the suspension may then be jet-cooked at a temperature between 95-140°C, preferably 100-135°C, such as 105-125°C, for about 1-15 minutes, preferably about 3-10 minutes. minutes, especially around about 5 minutes. The suspension is cooled to 60-95°C and more alpha-amylase variant and optional pullulanase variant and/or protease, preferably metalloprotease, are added to complete the hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at pH 4.0-6, in particular at pH 4.5 to 5.5. Saccharification step (b) can be carried out using conditions well known in the art. For example, a complete saccharification process can last from about 24 to about 72 hours, however, it is usual to do only a pre-saccharification of typically 40-90 minutes at a temperature between 30-65°C, typically about 60°C. C, followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF process). Saccharification is typically carried out at a temperature of 20-75°C, preferably 40-70°C, typically about 60°C, and at a pH between 4 and 5, usually about pH 4.5. The most widely used process for producing a fermentation product, especially ethanol, is a simultaneous saccharification and fermentation (SSF) process, in which there is no holding phase for saccharification, meaning that a fermenting organism can be added. , such as yeast, and enzyme(s). SSF can typically be carried out at a temperature of 25°C to 40°C, such as 28°C to 35°C, such as 30°C to 34°C, preferably about 32°C. In one embodiment, the fermentation is carried out for 6 to 120 hours, in particular 24 to 96 hours.

Glucoamylase Present and/or Added in Saccharification and/or Fermentation

The carbohydrate source-generating enzyme present during saccharification may, in one embodiment, be a glucoamylase. A glucoamylase is present and/or added in the saccharification and/or fermentation, preferably simultaneous saccharification and fermentation (SSF), in a process of the invention (ie, saccharification and fermentation of ungelatinized or gelatinized starch material).

In one embodiment, the glucoamylase present and/or added in the saccharification and/or fermentation is of fungal origin, preferably from an Aspergillus strain, preferably A. niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a Talaromyces strain, preferably T. emersonii or a Trametes strain, preferably T. cingulata, or a Pycnoporus strain, preferably P. sanguineus, or a Gloeophyllum strain, such as G. serpiarium or G. trabeum, or a strain of Nigrofomes.

In one embodiment, the glucoamylase is derived from Trametes, such as a Trametes cingulata strain, such as that shown in SEQ ID NO: 4 herein.

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 4 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 4 herein.

In one embodiment, the glucoamylase is derived from Talaromyces, such as a Talaromyces emersonii strain, such as that shown in SEQ ID NO: 5 herein.

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 5 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 5 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguineus described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as that shown as SEQ ID NO: 4 in WO 2011/066576.

In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 6 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 6 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strain of Gloeophyllum, as described in WO 2011/068803 (SEQ ID NO: 2 , 4, 6, 8, 10, 12, 14 or 16). In a preferred embodiment, the glucoamylase is Gloeophyllum sepiarium shown in SeQ ID No: 2 in WO 2011/068803 or SEQ ID NO: 7 herein.

In one embodiment, the glucoamylase is derived from Gloeophyllum serpiarium, such as that shown in SEQ ID NO: 7 herein. In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 7 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 7 herein.

In another embodiment, the glucoamylase is derived from Gloeophyllum trabeum such as that shown in SEQ ID NO: 8 herein. In one embodiment, the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 8 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 8 herein.

In one embodiment, the glucoamylase is derived from a strain of the genus Nigrofomes, in particular a strain of Nigrofomes sp. described in WO 2012/064351.

In one embodiment, glucoamylases can be added to the saccharification and/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2 AGU/g DS. Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from DuPont).

According to a preferred embodiment of the invention, the glucoamylase is present and/or added in the saccharification and/or the fermentation in combination with an alpha-amylase. Examples of suitable alpha-amylase are described below.

Alpha-Amylase Present and/or Added in Saccharification and/or Fermentation

In one embodiment, an alpha-amylase is present and/or added in the saccharification and/or fermentation processes of the invention. In a preferred embodiment, the alpha-amylase is of fungal or bacterial origin. In a preferred embodiment, the alpha-amylase is an acid-stable fungal alpha-amylase. A fungal acid-stable alpha-amylase is an alpha-amylase having activity in the pH range of 3.0 to 7.0 and preferably in the pH range of 3.5 to 6.5, including activity at a pH of approximately 4.0, 4.5, 5.0, 5.5 and 6.0.

In a preferred embodiment, the alpha-amylase present and/or added in the saccharification and/or fermentation is derived from a strain of the Rhizomucor genus, preferably a Rhizomucorpusillus strain, such as that shown in SEQ ID NO: 3 in WO 2013/006756, such as a Rhizomucor pusillus alpha-amylase hybrid that has an Aspergillus niger linker and starch-binding domain, such as that shown in SEQ ID NO: 9 herein, or a variant thereof.

In one embodiment, the alpha-amylase is present and/or added in the saccharification and/or fermentation is selected from the group consisting of:

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 9 herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g.

e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide of SEQ ID NO: 9 herein.

In a preferred embodiment, the alpha-amylase is a variant of the alpha-amylase shown in SEQ ID NO: 9 having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H Y141W; G20S Y141W; A76G Y141W; G128D Y141W; G128D D143N; P219C Y141W; N142D D143N; Y141W K192R; Y141W D143N; Y141W N383R; Y141W P219C A265C; Y141W N142D D143N; Y141W K192R V410A; G128D Y141W D143N; Y141W D143N P219C; Y141W D143N K192R; G128D D143N K192R; Y141W D143N K192R P219C; G128D Y141W D143N K192R; or G128D Y141W D143N K192R P219C (using SEQ ID NO: 9 for numbering).

In one embodiment, the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably described as SEQ ID NO: 9 herein, preferably with one or more of the following substitutions: G128D, D143N, preferably G128D D143N (using SEQ ID NO: 9 for numbering), and wherein the alpha-amylase variant present and/or added in the saccharification and/or fermentation is at least 75% of identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even more preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% identity to the polypeptide of SEQ ID NO : 9 in this memory.

In a preferred embodiment, the ratio of glucoamylase to alpha-amylase present and/or added during saccharification and/or fermentation may preferably be in the range of 500:1 to 1:1, such as 250:1 to 1:1. such as 100:1 to 1:1, such as 100:2 to 100:50, such as 100:3 to 100:70.

Starch-Containing Materials

Any suitable starch-containing starting material may be used in a process of the present invention. The starting material is generally selected based on the desired fermentation product. Examples of starch-containing starting materials suitable for use in the methods of the present invention include barley, lima beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat . and whole grains or any mixture thereof. The starch-containing material may also be a waxy or non-waxy type of corn and barley. In a preferred embodiment, the starch-containing material is corn. In a preferred embodiment, the starch-containing material is wheat.

Fermentation Products

The term "fermentation product" means a product produced by a method or process that includes fermentation using a fermenting organism. Fermentation products include alcohols ( eg, ethanol, methanol, butanol); organic acids ( eg, citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones ( eg, acetone); amino acids ( eg, glutamic acid); gases ( eg, H ² and CO ² ); antibiotics ( eg, penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, ^B12, beta - carotene..); and hormones. In a preferred embodiment, the fermentation product is ethanol, e.g. eg, fuel ethanol; drinkable ethanol, ie drinkable neutral spirits; o Industrial ethanol or products used in the consumable alcohol industry ( eg, beer and wine), dairy industry ( eg, fermented milk products), leather industry, and tobacco industry. Preferred types of beer include ales, stouts, porters, lagers, bitters, malt spirits, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In a preferred embodiment, the fermentation product is ethanol.

Fermenting Organisms

The term "fermenting organism" refers to any organism, including bacterial and fungal organisms, such as yeasts and filamentous fungi, suitable for producing a desired fermentation product. Suitable fermentation organisms are capable of fermenting, ie converting, fermentable sugars, such as arabinose, fructose, glucose, maltose, mannose or xylose, directly or indirectly to the desired fermentation product.

Examples of fermentative organisms include fungal organisms such as yeast. Preferred yeasts include strains of Saccharomyces, in particular Saccharomyces cerevisiae or Saccharomyces uvarum; Pichia strains, in particular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichia pastoris; Candida strains, in particular Candida arabinofermentans, Candida boidinii, Candida diddensii, Candida shehatae, Candida sonorensis, Candida tropicalis, or Candida utilis. Other fermentative organisms include strains of Hansenula, particularly Hansenula anomala or Hansenula polymorpha; Kluyveromyces strains, in particular Kluyveromyces fragilis or Kluyveromyces marxianus; and strains of Schizosaccharomyces, in particular Schizosaccharomyces pombe.

Preferred bacterial spoilage organisms include Escherichia strains, in particular Escherichia coli, Zymomonas strains, in particular Zymomonas mobilis, Zymobacter strains, in particular Zymobactorpalmae, Klebsiella strains in particular Klebsiella oxytoca, Leuconostoc strains, in particular Leuconostoc mesenteroides, Clostridium, in particular Clostridium butyricum, Enterobacter strains, in particular Enterobacteraerogenes, and Thermoanaerobacter strains, in particular Thermoanaerobacter BG1L1 (Appl. Microbiol. Biotech. 77: 61-86), Thermoanarobacter ethanolicus, Thermoanaerobacter mathranii, or Thermoanaerobacter thermosaccharolyticum. Strains of Lactobacillus are also envisioned, as well as strains of Corynebacterium glutamicum R, Bacillus thermoglucosidaisus, and Geobacillus thermoglucosidasius.

In one embodiment, the fermenting organism is a C6 sugar fermenting organism, such as a strain of, e.g. eg, Saccharomyces cerevisiae.

In one embodiment, the fermenting organism is a C5 sugar fermenting organism, such as a strain of, e.g. eg, Saccharomyces cerevisiae.

In one embodiment, the fermentation organism is added to the fermentation medium such that the count of viable fermentation organisms, such as yeast, per mL of fermentation medium is in the range of 105 to 1012, preferably 107 to 1010, especially approximately 5x107.

Yeast is the preferred fermenting organism for ethanol fermentation. Saccharomyces strains, especially strains of the species Saccharomyces cerevisiae, are preferred, preferably strains that are resistant to high levels of ethanol, ie up to, e.g. g., about 10, 12, 15, or 20% by vol. or more of ethanol.

In one embodiment, the yeast using C5 is a strain of Saccharomyces cerevisiae described in WO 2004/085627.

In one embodiment, the fermenting organism is a C5 eukaryotic microbial cell to which WO 2010/074577 (Nedalco) refers.

In one embodiment, the fermenting organism is a transformed C5 eukaryotic cell capable of directly isomerizing xylose to xylulose described in US 2008/0014620 .

In one embodiment, the fermenting organism is a C5 sugar fermenting cell described in WO 2009/109633.

Commercially available yeasts include LNF SA-1, LNF BG-1, LNF PE-2 and LNF CAT-1 (available from LNF Brazil), RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesattre, USA). , Fa LI (available from Fleischmann's Yeast, USA), Tresca yeast SUPERSTART and THERMOSACC™ (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA , USA), GERT STrAn D (available from Gert Strand AB, Sweden) and FERMIOL (available from DSM Specialties).

The fermentation organism capable of producing a desired fermentation product from fermentable sugars is preferably cultured under precise conditions at a particular growth rate. When the fermentation organism is introduced or added to the fermentation medium, the inoculated fermentation organism goes through a series of stages. Initially no growth occurs. This period is called the "lag rate" and can be considered an adaptation period. During the next phase, called the "exponential phase", the growth rate gradually increases. After a period of maximum growth, the rate ceases and the termentator organism enters the "steady rate". After an additional period of time, the fermenting organism enters "death rate" where the number of viable cells decreases.

Fermentation

The heating conditions are determined based on e.g. eg, on the type of plant material, the available heatable sugars, the heatable organism(s), and/or the desired heatable product. One skilled in the art can readily determine the proper heating conditions. The fermentation is can be carried out under the conditions of conventional use. Preferred fermentation procedures are anaerobic procedures.

For example, fermentations can be carried out at temperatures as high as 75°C, eg. eg, between 40-70°C, such as between 50-60°C. However, bacteria with a significantly lower optimum temperature down to around room temperature (around 20°C) are also known. Examples of suitable fermenting organisms can be found in the "Fermenting Organisms" section above.

For the production of ethanol using yeast, the fermentation can last from 24 to 96 hours, in particular from 35 to 60 hours. In one embodiment, the fermentation is carried out at a temperature between 20 and 40°C, preferably between 26 and 34°C, in particular around 32°C. In one embodiment, the pH is from 3 to 6, preferably around pH 4 to 5.

Other fermentation products can be fermented at temperatures known to the person skilled in the art, suitable for the fermenting organism in question.

Fermentation is typically carried out at a pH in the range of 3 to 7, preferably pH 3.5 to 6, such as around pH 5. Fermentations are typically carried out for 6-96 hours.

The processes of the invention can be carried out as a batch process or as a continuous process. The fermentations can be carried out in an ultrafiltration system in which the retentate is kept in recirculation in the presence of solids, water and the fermenting organism, and in which the permeate material is the liquid containing the desired fermentation product. Likewise, methods/procedures carried out in continuous membrane reactors with ultrafiltration membranes and in which the retained material is kept in recirculation in the presence of solids, water and the fermenting organism or organisms and in which the permeated material is the product are contemplated. fermentation containing liquid. After fermentation, the fermenting organism can be separated from the fermented suspension and recycled.

Fermentation Medium

The term "fermentation media" or "fermentation medium" refers to the environment in which the fermentation is carried out and includes the fermentation substrate, that is, the source of carbohydrates that is metabolized by the organism or organisms fermenters.

The fermentation medium may comprise other nutrients and a growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the fermentation art and include nitrogen sources such as ammonia; vitamins and minerals, or combinations thereof.

Recovery

After fermentation, the fermentation product can be separated from the fermentation medium. The fermentation medium can be distilled to extract the desired fermentation product or the desired fermentation product can be extracted from the fermentation medium by microfiltration or membrane filtration techniques. Alternatively, the fermentation product can be recovered by extraction. Recovery methods are well known in the art.

The present invention is further described by the following examples.

EXAMPLES

Example 1: Cloning and expression of wild-type Meripilus giganteus protease III in A soeraillus oryzae

The cloning and expression of wild-type serine protease III from Meripilus giganteus (MgP3) belonging to the S53 family was described in WO 2014/037438. The cloned gene was subsequently transferred as a polymerase chain reaction (PCR) product with suitable pendants for insertion into P_BGMH0016, described in WO2013/119302 (example 18 and figure 10), by cloning USER™ ( New England Biolabs, Ipswich, MA, USA) for expression in Aspergillus oryzae. Plasmid DNA was used as a template in the PCR reaction. The forward primer sequence was 5'-AGAGCGAUaccatggtcgccaccagc-3' (SEQ ID NO: 11), and the reverse primer sequence was 5'-AACGTCACGUtcacaggccgaccgcggt-3' (SEQ ID NO: 12). Phusion U DNA polymerase (Thermo Fischer Scientific, Grand Island, NY, USA) was used for the PCR reaction

The PCR reaction consisted of: 10 ng template DNA, 0.5 pM each of the primers, 1 U Phusion U polymerase, 0.25 mM dNTPs, 1x Phusion HF buffer, water to 50 pl. The PCR program consisted of an initial denaturation for 30 seconds at 98°C, 35 cycles of amplification (20 seconds at 98°C; 20 seconds at 60°C; 2 minutes at 72°C) and a final extension of 5 minutes. at 72°C.

The amplified gene was purified (QIAquick, QIAGEN) and inserted into P_BGMH0016 by USER™ cloning. The reaction contained: 100ng P_BGMH0016 vector as a PCR product, 500ng purified PCR product, 1gl USER™ enzyme, 1gl Phusion HF buffer, water to 10gl. The program consisted of 25 minutes at 37°C and 25 minutes at 25°C. The mixture was transformed into 50 gl of chemically competent Escherichia coli TOP10 (DH10p) cells, selected for their resistance to ampicillin, and the correct clones were verified by sequencing. The plasmid was transformed into Aspergillus oryzae Cols1300 protoplasts described in WO2013/119302, selected by growth on nitrate as sole nitrogen source, and correct clones were verified by Sanger sequencing. The use of the BGMH0016 plasmid

takes advantage of the so-called double split marker system (DSMS) or bi-partite selection marker system (Nielsen et al. 2006, (Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans, Fungal Genetics and Biology, 2006(43 ) 54 to 64)). The double-cleaved marker system in pBGMH0016 consists of two double-cleaved markers in Aspergillus oryzae strain COLS1300: niaüú and niiAú, which flank the amdS selection marker. Only correct insertion of donor DNA via double homologous recombination into the partial niaD and niiA genes, respectively, in the COLS1300 host cell will restore the functionality of both genes, and both genes are required for the cell to grow in a medium that contains sodium nitrate as the sole source of nitrogen. This also ensures a targeted insert instead of the amdS gene in a single copy.

Each of the specific variants of MgP3 according to the invention can be cloned and expressed as described above.

Example 2: Stability test for specific variants according to the invention

The stability of MgP3 substitution variants relative to MgP3 having the amino acid sequence of SEQ ID NO: 3 is determined by incubating the protease samples under defined conditions ("stress conditions") in a buffered solution. The temperature and duration of incubation are chosen such that the remaining wild-type activity after incubation is equal to approximately 20% of the activity of a similar sample incubated under defined conditions ("reference conditions") that do not lead to loss of activity when incubated for the same duration. Activity after incubation under stress or reference conditions is determined using the Suc-AAPF-pNA assay described below.

A. Determination of protease activity using the Suc-AAPF-pNA assay

In order to determine the protease activity of the MgP3 protease and variants thereof, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-propyl-L-phenyl-p-nitroanilide (Suc- AAPF-pNA).

The reagent solutions used are:

Dilution Buffer: 100 mM succinic acid, ^{1 mM CaCl 2} , 150 mM KCl, 0.01% Triton X-100, pH 4.5. Assay Buffer: Dilution buffer with Suc-AAPF-pNA (1 mM N-succinyl-L-alanyl-L-alanyl-L-propyl-L-phenyl-p-nitroanilide, Bachem 4002299.1000), 12.5 gL mL-1 DMSO (Amresco 0231).

MgP3 variants were grown in 96-well microtiter plates to produce culture supernatant. In each of the plates, four wells contained wild-type MgP3 culture supernatant (SEQ ID NO: 3).

The Suc-AAPF-pNA assay is performed in 384-well flat bottom polystyrene disposable microplates (Perkin-Elmer 6007649). First, protease samples (culture supernatant) are diluted 5-fold in Dilution Buffer. Subsequently, 25 gL of the diluted protease sample is added to each well of the 384-well assay microplate, followed by the addition of 25 gL of Assay Buffer. The solutions are mixed thoroughly and the absorbance recorded at 405nm for 5 minutes, and the initial rate per well calculated from the maximum rate of a 270 second window.

B. Stability test

The stability of the MgP3 variants in buffer is determined by determining the stability half-life of the protease variants as the negative value of the stress time in hours, divided by the logarithm to base 2 of the ratio of the protease activity of the variants under unstable conditions. ("stress conditions") to the protease activity of the same variants under non-destabilizing conditions ("reference conditions"). Kinetic slopes were quality controlled and variant samples were removed that 1) showed a linear fit R2 that was below the 25% quartile of the wild-type control R2 fit in a) stressed condition or b) unstressed condition , or 2) showed Vmax below the 75% quartile of Vmax from non-expressing controls, in a) the stressed condition or b) the unstressed condition. The ratio of the initial rate of stressed and unstressed supernatants originating from the same clone was defined as

Vmax Stressed

Ratio =

Vmax Unstressed

Samples showing a ratio above the 90% quantile of the wild-type control were counted as hits, and were harvested, recultured, and sequenced. _{The half-life at 60°C} ⁱ _v ^t ₅ ^V ₀ ^¿ . _c ) _{/ was calculated based on} the

Ln (0.5)

L 7 l ( Ratio)^

5 ' equation, where half-life is given in minutes.

The reagent solutions used are:

Dilution Buffer: as described in A.

Assay Buffer: as described in A.

The assay is performed in 96-well polystyrene disposable PCR plates (eg, Thermo Scientific AB-0601), except for the Suc-AAPF-pNA assay described in A, which is performed in 384-well microplates.

First, 25 pl of the culture supernatants containing the protease are transferred to two new plates: one designated "Stress Plate" and one designated "Reference Plate". The Stress Plate is fitted with a lid and is incubated in a thermocycler for 5 minutes at 60°C. The Reference Plate is provided with a lid and meanwhile incubated at 21°C (room temperature). After incubation, culture supernatants from the Stress Plate and Reference Plate are transferred to 384-well plates and 25 pL of assay buffer is added to each well of the Stress Plate and Reference Plate. Reference, and mix well.

Half-Life Enhancement Factor (HIF)

The half-life enhancement factor (HIF) correlates the Stability Half-Life of a variant protease with that of the reference protease. The calculation of the improvement factor based on the Stability Half-life (SH) was performed according to the following equation: HIF = (SH of the variant) / (SH of MgP3 (SEQ ID NO: 3)).

A half-life enhancement factor greater than 1 (HIF > 1) indicates improved stability of a variant compared to the reference, while a HIF of 1 (HIF = 1) identifies a variant that is on par with the reference . and an HIF of less than 1 (HIF < 1) identifies a variant that is less stable than the reference. E1HIF of all mutations with enhanced stability is shown in Table 1 below. If a given substitution was observed more than once, the median HIF of all observations is shown.

Table 1

Example 3: Specific activity assay for specific variants according to the invention

The specific activity of the MgP3 substitution variants relative to MgP3 having the amino acid sequence of SEQ ID NO: 3 is determined by comparing the total activity of the protease samples to the amount of protein in the sample. Activity in culture supernatants is determined using the Suc-AAPF-pNA assay and protein concentration determined by enzyme-linked immunosorbent assay (ELISA), both described below.

A. Determination of protease activity using the Suc-AAPF-pNA assay

In order to determine the protease activity of the MgP3 protease and variants thereof, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-propyl-L-phenyl-p-nitroanilide (Suc- AAPF-pNA).

The reagent solutions used are:

Dilution Buffer: 100 mM succinic acid, ^{1 mM CaCl 2} , 150 mM KCl, 0.01% Triton X-100, pH 4.5. Assay Buffer: Dilution buffer with Suc-AAPF-pNA (1 mM N-succinyl-L-alanyl-L-alanyl-L-propyl-L-phenyl-p-nitroanilide, Bachem 4002299.1000), 12.5 pL mL-1 DMSO (Amresco 0231).

MgP3 variants were grown in 96-well microtiter plates to produce culture supernatant. In each of the plates, four wells contained wild-type MgP3 culture supernatant (SEQ ID NO: 3). The Suc-AAPF-pNA assay is performed in 384-well flat bottom polystyrene disposable microplates (Perkin-Elmer 6007649). First, protease samples (culture supernatant) are diluted 5-fold in Dilution Buffer. Subsequently, 25 pL of the diluted protease sample is added to each well of the 384-well assay microplate, followed by the addition of 25 pL of Assay Buffer. The solutions are mixed thoroughly and the absorbance recorded at 405nm for 5 minutes, and the initial rate per well calculated from the maximum rate of a 270 second window.

B. Protein quantification by enzyme-linked immunosorbent assay (ELISA)

The reagent solutions used are:

TBS-T: 20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5.

PBS buffer: 137 mM NaCl, 2.7 mM KCI, Na2HPO410 mM KH ² PO ⁴ 1.8 mM

TMB plus 2 (KemEnTech diagnostics A/S) is prefabricated and ready for use.

The protease sample (culture supernatant) was diluted 2000-fold in TBS-T and analyzed by ELISA, using a rabbit-produced HRP-conjugated MgP3 antibody. ELISA quantitation is carried out in a blocked 384 microtiter plate (Nunc MaxiSorp) coated with "protein A" purified rabbit anti-MgP3 polyclonal antibodies. Diluted enzyme samples (as well as standards and controls) are transferred to the antibody plate, incubated, and then repeatedly washed vigorously with PBS buffer. Detection is carried out using the same polyclonal antibodies, but in an HRP-labelled form (LL-HRP conjugation). Anti-MgP3:HRP conjugate in TBS-T is added at the appropriate dilution, followed by washing with PBS. The signal is read as the OD-endpoint (620 nM) after the addition of TMB plus 2 substrate.

Concentrations were calculated using a nonlinear function that was derived from the standard MgP3-containing samples that were placed in external plates, as well as from the standard MgP3 wells that were added to each of the plates in the flow. tracking.

C. Calculation of specific protease activity for variants

A linear fit per plate was calculated based on the initial rate and standard concentrations of the standard samples. Plaques outside the 2.5-97.5% quantile range for the slope and intercept fit parameters were removed. A concentration prediction was derived from the Vmax by plugging it into the inverted linear fit of the standards (per plate). The specific activity was calculated as

Concentration (Predicted)

Specific activity =

Concentration [ELISA}

For the selection of hits, only the samples that showed an ELISA concentration < 30 pg mL-1 were considered. Samples showing specific activity above the 95% quantile of the wild-type control were counted as hits, and sequenced.

Activity Specific Enhancement Factor (AIF)

The specific activity enhancement factor (AIF) correlates the specific activity of a variant protease with that of the reference protease. The calculation of the enhancement factor based on the specific activity (SA) was performed according to the following equation: AIF = (SA of the variant) / (SA of MgP3 (SEQ ID NO: 3)).

A specific activity enhancement factor greater than 1 (AIF > 1) indicates improved specific activity of a variant compared to the reference, while an AIF of 1 (AIF = 1) identifies a variant that is on par with The reference. and an AIF of less than 1 (AIF < 1) identifies a variant with a lower specific activity than the reference. The AIF of all mutations with enhanced specific activity is shown in Table 2 below. If a given substitution was observed more than once, the median AIF of all observations is shown.

Table 2

Example 4: Determination of the expression level for specific variants according to the invention The expression levels of substitution variants of MgP3 with respect to MgP3 having the amino acid sequence of SEQ ID NO: 3 are determined by the total amount of protein MgP3 in the culture supernatant. Using a system where precisely one copy of the variant is integrated at a defined locus, and that locus is identical for all variants, allows direct comparison of expression levels between specific variants. Such a system could be the DSMS system described in Example 1. Protein concentration was determined by enzyme-linked immunosorbent assay (ELISA), described below.

TO.

Protein quantification by enzyme-linked immunosorbent assay (ELISA)

The reagent solutions used are:

TBS-T: 20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5.

PBS buffer: 137 mM NaCl, 2.7 mM KCI, Na2HPO410 mM KH ² PO ⁴ 1.8 mM

TMB plus 2 (KemEnTech diagnostics A/S) is prefabricated and ready for use.

The protease sample (culture supernatant) was diluted 2000-fold in TBS-T and analyzed by ELISA, using a rabbit-produced HRP-conjugated MgP3 antibody. ELISA quantitation is carried out in a blocked 384 microtiter plate (Nunc MaxiSorp) coated with "protein A" purified rabbit anti-MgP3 polyclonal antibodies. Diluted enzyme samples (as well as standards and controls) are transferred to the antibody plate, incubated, and then repeatedly washed vigorously with PBS buffer. Detection is carried out using the same polyclonal antibodies, but in an HRP-labelled form (LL-HRP conjugation). Anti-MgP3:HRP conjugate in TBS-T is added at the appropriate dilution, followed by washing with PBS. The signal is read as the OD-endpoint (620 nM) after the addition of TMB plus 2 substrate.

Concentrations were calculated using a nonlinear function that was derived from the standard MgP3-containing samples that were placed in external plates, as well as from the standard MgP3 wells that were added to each of the plates in the flow. tracking.

Expression level enhancement factor (EIF)

The expression level enhancement factor (EIF) correlates the expression level of a variant protease with that of the reference protease. Calculation of the enhancement factor EIF based on expression level (EL) was performed according to the following equation: ID = (EL of variant) / (EL of MgP3 (SEQ ID NO: 3)).

An expression level enhancement factor that is greater than 1 (EIF > 1) indicates an improved expression level of a variant compared to the reference, while an EIF of 1 (EIF = 1) identifies a variant that is less than the reference. par with the reference. and an EIF of less than 1 (EIF < 1) identifies a variant that has a lower expression level than the reference. The EIF of all mutations with enhanced expression levels is shown in Table 3 below. If a given substitution was observed more than once, the median EIF of all observations is shown.

Table 3

Example 5: Variants Having Increased Thermostability As Determined By Residual Activity For Specific Combinations Of Substitutions After Thermal Stress

Based on the data obtained for single substitution variants according to the invention, several variants that have multiple substitutions and show an increase in residual activity after incubation at selected temperatures in the range of 56°C to 70°C for 20 minutes

Variants harboring substitutions compared to wild-type MgP3 (SEQ ID NO: 3) as shown in Table 4, were constructed and expressed in A. oryzae. A. oryzae MgP3 strains harboring and expressing the MgP3 combination variants were grown in microtiter plates (MTP) in MDU-2 (225 pL/well MTP) for expression, for 5 days at 30°C. in 96-well capped MTP without shaking. Expression plates also contained wild-type control MgP3 expressed in A. oryzae. Stringy material was removed by pressing a Biopress® plate down into the wells. The supernatant was diluted in assay buffer (100 mM succinic acid, mM CaCh1, 150 mM KCl, 0.01% Triton X-100, pH 4.5). A fraction of the diluted supernatant was transferred to a 96-well thermal cycler plate. The 96-well plate was transferred to a TRobot® thermocycler (Biometra) and heated at the temperatures noted in Table 4 (56°C, 60°C, or 70°C) for 20 min, followed by cooling to 25°C. C for 2 min. Volumes (25 pL) of the stressed and unstressed supernatant were transferred to a clear 384-well MTP plate. Assay solution (25 pL, assay buffer, with 1 mM succinyl-AAPF-pNA, 12.5 pL mL-1 DMSO) is dispensed into the 384-well MTP. Absorbance at 405 nm was recorded for 10 min and the initial rate per well was calculated from the maximum rate of a 9 min window. The ratio of the initial rate of stressed and unstressed supernatants originating from the same clone was defined as

Vmax Stressed

Ratio =

Vmax Unstressed

which translates into residual activity (%), which is shown in Table 4.

Claims (27)

1. A protease variant comprising a substitution at one or more positions corresponding to the positions 1, 5, 9, 25, 29,30, 32, 38, 39, 40, 41, 42, 45, 46, 49, 50, 53, 54, 55, 57, 59, 60, 61, 62, 64 , 67, 68, 69, 74, 76, 77, 78, 79, 80, 81, 82, 87, 93, 94, 96, 99, 102, 103, 105, 109, 111, 112, 114, 115, 117, 122, 123, 124, 128, 130, 136, 141, 142, 145, 146, 147, 150, 153, 154, 157, 159, 161, 167, 169, 175, 182, 185, 199, 200, 205, 207, 209, 210, 213, 216, 217, 225, 228, 231, 234, 237, 242, 244, 245, 246, 248, 258, 262, 266, 267, 269, 271, 272, 276, 278, 280, 284, 289, 293, 296, 299, 305, 308, 313, 314, 317, 318, 319, 321, 322, 324, 325, 326, 329, 330, 331, 333, 336, 338, 341, 343, 345, 347, 348, 355, 358, 359, 361, 362, 363, and 364 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, and wherein the variant has increased thermostability measured as enhancement factor, HIF, compared to with the protease of SEQ ID NO: 3, wherein the variant comprises at least one substitution selected from the group consisting of: A1T, S5I, S5K, S5L, S5Y, T9H, K25P, S29I, S29P, S29R, S30E, K32W, F38L, 139G, I39L, I39V, I39Y, D40F, D40G, D40H, D40N, D40P, Q41I, Q41V, F42C, F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, A46E, K49P, S50C, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54I, Q54L, Q54M, Q54V, F55H, F55L, K57P, K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C, E74H, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A, E81H, E81K, E81L, E81P, E81Y, A82F, A82L, A82M, A82P, N87S, T93L, T93S, V94S, L96W, G99C, T102L, T102V, T103I, T103K, T103V, I105Q, D109H, D109K, D109M, D109Y, F111H, F111K, F111S, F111Y, Q112R, G114L, N115A, N115C, N115K, N115P, N115R, N115T, N115Y, E117D, E117G, E117H, E117N, E117Q, E117S, I122K, I122R, I123A, N124L, N124V, G128A, G128E, G128F, G128L, G128S, S130D, S130G, S130L, S130N, S130P, L136K, G141T, Q142A, Q142F, Q142L, Q142N, N145V, T146A, T146I, T146L, T146N, I147S, K150F, K150L, K150R, N153A, N153L, N153M, N153P, N153R, N153Y, Q154A, Q154I, Q154L, Q154M, Q154N, Q154T, Q154V, N1571, N157L, Y159H, Q161E, T167F, I169F, I169S, D175I, D175N, Q182C, Q182T, Q182V, H185P, F199L, F199M, M200S, A205S, Q207H, Q207L, V209F, S210T, T213L, T213R, A216R, F217I, F217Y, V225Y, I228L, I228T, I228W, Y231I, S234Q, S237F, S237Q, A242I, A242L, A242V, G244S, S245P, T246S, S248Y, F258P, S262P, V266K, V266R, D267A, D267H, D267L, D267N, Q269L, V271R, S272I, S272K, S272L, S272R, S272T, S272V, S272Y, T276L, T276Y, G278P, D280N, D280S, D280T, D280Y, C284A, C284G, F289Y, I293T, V296A, V296I, R299N, K305S, L308V, P313L, F314I, F314L, S317E, S317N, S317W, S318P, A319F, A319Q, K321L, K321Q, K321S, A322D, L324Q, L324S, N325L, D326P, S329C, S329H, S329I, S329L, S329N, S329T, S329V, S329W, G330Q, S331W, S331Y, P333R, S336K, S336N, N338M, N338Q, N338R, P341S, K343A, K343H, K343S, K343T, K343V, G345S, D347I, P348Y, P355I, P355R, A358L, A358P, A358R, A358Y, K359L, K359S, L361M, T362L, T362N, A363G, A363L, and V364I, and wherein the substitution at the one or more positions provides a protease variant having a increase in thermostability measured as enhancement factor, HIF, of at least 1.4. 2. The variant of claim 1, comprising at least one substitution selected from the group consisting In : S5K, S5L, S5Y, T9H, K25P, S29R, S30E, K32W, F38L, I39G, I39L, I39V, I39Y, D40F, D40G, D40H, D40N, D40P, Q411, Q41V, F42C, F42K, F42S, F42Y, K45L, K45M, K45Y, A46C, K49P, S50I, S50L, S50M, S50Q, A53C, A53E, A53V, Q54I, Q54L, Q54M, Q54V, F55H, F55L, K57P, K57S, I59G, I59M, I59P, S60P, S61L, S61P, S62C, S62N, S62P, T64G, T64I, L67V, Q68V, Q68Y, T69C, E74R, D76L, D76R, Q77L, S78D, P79Y, S80L, E81A, E81H, E81K, E81L, E81P, E81Y, A82M, A82P, N87S, T93L, V94S, G99C, T102L, T102V, T103I, T103K, T103V, I105Q, D109H, D109K, D109M, D109Y, F111H, F111K, F111S, Q112R, G114L, N115C, N115K, N115R, N115T, N115Y, E117D, E117G, E117H, E117N, E117Q, E117S, I122R, N124L, G128F, G128S, S130G, S130N, S130P, L136K, G141T, Q142A, Q142F, Q142L, Q142N, N145V, T146A, T146L, T146N, I147S, K150L, N153M, N153P, N153R, Q154I, Q154L, Q154N, Q154T, Q154V, N157L, Q161E, T167F, I169F, D175N, Q182C, Q182T, Q182V, F199L, F199M, M200S, A205S, Q207H, Q207L, V209F, S210T, T213R, F217I, V225Y, I228L, I228T, I228W, S234Q, S237F, G244S, S245P, T246S, S248Y, F258P, S262P,V266K, V266R, D267A, D267H, D267L, D267N, Q269L, V271R, S272K, S272L, S272R, S272V, S272Y, T276Y, G278P, D280N, D280S, D280T, C284G, F289Y, I293T, V296A, R299N, K305S, L308V, F314L, S317E, S317W, S318P, A319F, A319Q, K321L, K321Q, K321S, A322D, L324Q, L324S, D326P, S329C, S329H, S329I, S329L, S329N, S329W, G330Q, S331W, S331Y, S336K, S336N, P341S, K343A, K343H, K343S, K343T, G345S, D347I, P348Y, P355I, A358L, A358P, A358R, A358Y, K359L, K359S, L361M, T362L, A363G, A363L, and V364I, and where the substitution at one or more positions provides a protease variant that has an increase in thermostability measured as enhancement factor, HIF, of at least 1.6. 3. A protease variant comprising a substitution at one or more positions corresponding to the positions 20, 27, 29, 34, 40, 42, 45, 46, 50, 53, 54, 55, 58, 59, 60, 61, 62, 66, 69, 76, 81, 87, 93, 96, 98 , 101, 102, 103, 109, 110, 115, 117, 122, 124, 125, 127, 128, 130, 136, 141, 142, 145, 146, 148, 149, 150, 153, 154, 157, 159, 161, 164, 167, 182, 183, 184, 185, 188, 199, 200, 207, 208, 209, 210, 211, 213, 216, 219, 228, 231, 252, 253, 261, 266, 267, 272, 275, 276, 277, 280, 285, 288, 291, 292, 293, 294, 296, 299, 303, 308, 310, 317, 318, 319, 321, 326, 327, 336, 338, 341, 344, 345, 348, 356, 358, 359, 361, 362, 363, and 364 polypeptide of SEQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identity of the sequence with the mature polypeptide of SEQ ID NO: 3, wherein the variant comprises at least one substitution selected from the group consisting of: G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, D40S, D40Y, F42Y, K45L, K45Y, A46E, A46K, S50A, S50C, S50I, S50L, S50T, A53I, A53K, A53L, A53S, Q54L, F55R, D58M, I59M, I59P, S60C, S60E, S60P, S61D, S61P, S62C, S62N, S66F, S66L, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P, D76V, D76Y, E81S, N87F, T93S, L96F, L96S, L96T, T98L, P101F, P101L, P101M, P101R, T102C, T102I, T103V, D109P, D110W, N115P, E117D, E117F, E117G, E117L, E117W, E117Y, I122L, N124D, N124I, N124K, N124L, N124M, N124T, N124V, F125K, F125L, F125M, L127C, L127P, G128W, G128Y, S130D, S130F, S130I, S130N, L136C, G141A, G141C, G141F, G141L, G141M, G141Q G141R, G141V, Q142A, Q142L, Q142M, Q142S, Q142W, N145F, N145G, N145I, N145L, N145P, N145R, N145S, N145V, N145W, T146A, T146C, T146H, T146I, T146M, T146N, T146Q, T146R, T146V, T146W, S148G, A149V, K150F, N153D, N153F, N153G, Q154C, Q154F, Q154G, Q154L, Q154M, Q154L, Q154V, Q154T, Q154V, Q154W, N157F, N157A, N157E, N157F, N157A, N154E N157Y, Y159F, Y159G, Y159H, Y159S, Q161A, A164L, T167D, T167V, Q182F, S183A, S183F, A184F, A184H, A184I, A184K, A184L, A184Q, A184R, A184S, A184T, A184V, A184W, A184Y, H185A, H185L, H185P, H185S, N188L, N188Q, M200L, Q207F, G208H, V209F, V209G, V209W, V209Y, S210E, S210L, S210R, S210T S210Y, P211 F, T213F, T213H, T213i, T213L, T213N, T213R, T216F, A216L, S219m, I228F, I228H, I228L, I228M, I228Q, 1228V, I228Y, Y231F, Y231S, S237F, S237I , S237K, S237L, G244L, G244N, G244P, G244R, G249C, N252F, N252K, N252S, R253C, V261A, V261G, V266C, V266R, D267S, S272I, S272K, S272L, S272M, S272Q, S272R, S272T, S272Y, Q275A , Q275F, Q275K, Q275L, Q275R, Q275V, T276F, T276R, I277V, D280F, D280H, D280T, A285L, A285S, T288C, T288I, T288V, S291V, V292C, I293M, I293Y , S294T, S294V, V296L, R299P, A303C, A303F, A303H, A303S, A303V, G304H, G304K, G304P, G304R, G304S, G304Y, K305C, K305F, K305 H, K305I, K305L, K305M, K305S, K305T, K305Y, S306L, S306M, S306R, P307C, L308P, L308T, F310M, F310P, F310Y, S317A, S317C, S317G, S317H, S317W, S317LK, S317LK, S317Y, S318N, S318R, A319F, A319W, K321R, K321S, D326P, S336R, N338H, N338S, N338T, P3411, P341N, P341 R, A344K, A344L, A344P, G345K, G345S, P348G, P348L, P348S , N356L, N356Y, A358F, A358L, A358P, K359C, K359L, K359S, K359W, L361E, L361M, L361P, L361R, L361S, L361T, L361V, T362A, T362C, T362H, T362I, T362K, T362P, T362Q, T362R, T362V, T362Y, A363E, A363F, A363L, A363M, A363V, V364F, V364i, V364L, V364M, and V364S, and wherein substitution at one or more positions provides a protease variant having a increase in specific activity measured as enhancement factor, AIF, of at least 1.37. 4. The variant of claim 3, comprising at least one substitution selected from the group consisting of: G20L, G20R, T27S, S29A, S29C, S29L, S29N, S29P, S29R, A34L, A34N, A34R, F42Y, K45L, K45Y, A46E, A46K, S50A, S50L, A53L, Q54L, F55R, I59M, I59P, S60E, S60P, S61D, S61P, S62N, S66M, S66T, T69C, D76A, D76G, D76K, D76L, D76N, D76P, D76V, E117F E117L, E117N, E117P, E117S, E117W, E117Y, N124D, N124M, N124T, N124V, F125K, F125L, F125M, L127C, L127P, G128N, G128R, G128S, G128W, G128Y, S130D, S130F S130i, S130N, L136C, G141A, G141C, G141M, G141L, G141R, G141V, Q142A, Q142L, Q142M, Q142S, Q142W, N145L, N145P, N145R, N145S, N145V, N145W, N145V, N145W, N145V, N145W T146A, T146C, T146H, T146I, T146M, T146N, T146Q, T146R, T146V, T146W, S148G, A149V, K150F, N153Y, Q154I, Q154K, Q154L, Q154M, Q154R, Q1 54W, N157F, N157Y, Y159H, Y159S, Q161A, T167D, Q182F, A184F, A184I, A184W, H185A, N188L, F199L, M200F, M200L, Q207F, G209G, V209H, V209L, V209G V209N, V209W, V209Y, S210E, S210L, S210R, S210T, S210Y, P211F, T213L, T213N, T213R, T213S, T213W, A216F, S219M, I228F, I228H, I228L, I228M, I228Q, I228V, I228Y Y231F, Y231S, S237F, S237I, S237K, S237L, G244N, G249C, N252F, V261A, V261G, V266C, V266R, D267S, S272I, S272L, S272M, S272Q, S272R, S272T, S272Y, Q275F, Q275K, Q275L, Q275R, Q275V, T276F, T276R, T276V, I277L, I277V, D280F, D280H, D280T, A285S, T288C, T288V, I293Y, S294T, S294V, A303F, G304H, G304K, G304P, G304R, G304S, K305C, G304S K305F, K305H, K305i, K305S, K305T, S306L, S306R, P307C, L308P, L308T, F310M, S317C, S317G, S317H, S317R, S317L, S317R, S317W, S317Y, S318N, S318R, S318N, S318R, S318N, S318R A319F, A319W, K321R, K321S, D326P, V327C, V327T, S336R, N338H, N338S, N338T, P3411, P341N, P341R, A344K, A344L, A344P, G345K, G3 45S, P348G, P348L, P348S, N356L, N356Y, A358L, K359S, K359W, L361 P, L361 R, T362C, T362H, T362I, T362K, T362M, T362R, T362Y, A363F, and V364M, and where the substitution in the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 2.0. 5. The variant of claim 3, comprising at least one substitution selected from the group consisting of: A34N, A34R, D76A, D76K, D76L, D76N, D76Y, N87F, E117F, E117G, E117L, E117N, E117P, E117W, Where the replacement at the one or more positions provides a protease variant having an increase in specific activity measured as enhancement factor, AIF, of at least 4.5. 6. The variant of any of claims 1-3, wherein the variant comprises a substitution selected from the group consisting of: I39L, K57P, I59M, S66T, T102V, F111H, N115K, D267N, S272Y, Q275K, D280S. 7. The variant according to claim 6, wherein the variant has increased thermostability measured as residual activity after thermal stress for 20 min at 56°C. 8. The variant according to claim 7, wherein the residual activity is at least 50%, in particular at least 60%, at least 70%, at least 80%. 9. The variant of any of claims 6-8, wherein the variant comprises at least three substitutions selected from the group consisting of: I39L, K57P, I59M, S66T, T102V, F111H, N115K, D267N, S272Y, Q275K, D280S . 10. The variant of claim 9, comprising the substitutions I39L+S66T+T102V or D267N+S272Y Q275K+D280S. 11. The variants of claim 10, comprising the substitutions: 139L+S66T T102V; 139L+S66T+T102V+D267N S272Y+D280S; I39L+K57P+I59M+S66T+T102V F111H+N115K+D267N+S272Y, 139L+K57P+I59M+S66T+T102V+F111H+N115K+D280S; or D267N+S272Y+Q275K+D280S. 12. A protease variant comprising at least one substitution corresponding to positions 2, 17, 22, 41, 41, 42, 45, 46, 54, 55, 57, 59, 60, 61, 62, 63, 64, 66, 69, 80, 82, 84, 87, 98, 99, 102, 103, 112, 114, 115, 129, 131, 134, 149, 159, 167, 169, 199, 200, 205, 207, 209, 211, 213, 217,242, 250, 266, 267, 269, 270, 271, 272, 274, 278, 279, 280, 284, 288, 291,292, 294, 296, 301, 307, 314, 329, 3,363 362, and 363 of the polypeptide of SEQ ID NO: 3, and the variant has protease activity, and wherein the variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO: 3, and wherein the variant comprises a substitution at one or more positions selected from the group consisting of: I2L, A17P, P22V, Q41L, Q41T, Q41V, Q41Y, F42R, K45S, A46L, A46W, Q54R, Q54S, Q54V, Q54Y, F55L, F55N, F55T, F55Y, K57H, K57L, K57V, I59D, I59K, I59R, I59S, I59V, S60I, S60L, S60Q, S 60R, S60Y, S61A, S61F, S62H, S62I, S62L, S62R, S62V, T63A, T63E, T63G, T63R, T63V, T64C, T64D, T64M, S66G, T69A, T69F, T69P, T69R, T69Y, S80G, S80L, S80N, S80T, A82H, I84G, N87P, T98C, G99V, T102A, T102H, T103F, T103I, T103V, Q112H, Q112M, Q112R, Q112S, G114A, G114C, G114L, G114P, N115Y, E129VS, E129VS, E129VS, E129VS, E129VS, E129VS, E129VS, E129VS, E129VS N131i, N131S, N131V, Q134A, Q134L, Q134R, A149D, Y159F, Y159L, T167A, T167L, T167S, T167W, I169S, Q207N, V209P, P211 F, P211S, T216, F217i, A242E, K250R I271I V271M, V271S, V271Y, S272A, S272N, G274R, G278S, V279I, D280Y, T288C, T288G, S291V, V292S, S294G, V296P, V296R, V296T, 1301T, P307T, F314L, S329D, S329V, S331A, S329D, S329V, S331A S331G, S331T, P333V, S336G, S336L, S336T, T362R, and A363T, and wherein substitution at one or more positions provides a protease variant having an au increase in expression level in Aspergillus oryzae measured as enhancement factor, EIF, of at least 1.38. 13. The variant of claim 12, comprising at least one substitution selected from the group consisting of: I2L, A17P, P22V, Q41L, Q41Y, F42R, Q54R, Q54S, Q54Y, F55N, F55T, K57L, K57V, I59D, I59S, I59V, S60L, S60Q, S60R, S60Y, S61F, S62L, S62V, T63A, T63E, T63R, T64C, T64D, T64M, Q112H, Q112M, Q112R, Q112S, G114A, G114C, G114L, G114P, E115LY, E115LY, N115LY, E129S, E129V, E129y, N131i, N131S, N131V, Q134R, A149D, T167A, T167S, T167W, F19y, A242E, K250R, V266A, V266F, V266L, V266S, V266T, V266Y, D267F, D267L, D267T D267V, Q269C, Q269F, Q269I, Q269S, Q269T, Q269V, Q269X, I270A, I270C, I270L, I270S, V271F, V271I, V271K, V271M, S272A, G274R, G278S, T288A, T288C, T288G, V296P, V296R, V296T, F314L, S329D, S329V, S331A, S331G, S331T, P333V, S336G, S336L, and S336T, and wherein the substitution at the one or more positions provides a protease variant that has an increased expression level measured as a factor of improvement, EIF, of at least 1.6. 14. The variants of any of claims 1-13, having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. 15. The variant of any of claims 1-14, wherein the number of substitutions is 1-20, e.g. eg, 1-10 and 1-5, such as 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. 16. A polynucleotide encoding the variant of any of claims 1-15. 17. A nucleic acid construct comprising the polynucleotide of claim 16. 18. An expression vector, comprising the polynucleotide of claim 16. 19. A recombinant host cell comprising the polynucleotide of claim 16. 20. A method of producing a protease variant, comprising: culturing the host cell of claim 19 under conditions suitable for expression of the variant and, optionally, recovering the variant. 21. A composition comprising a variant of any of claims 1-15. 22. The composition of claim 21, further comprising a glucoamylase and optionally a fungal alpha-amylase. 23. A process for preparing a fermentation product from starch-containing material, comprising simultaneously saccharifying and fermenting starch-containing material using enzymes that generate a carbohydrate source and a fermenting organism at a temperature below initial gelatinization temperature of said starch-containing material in the presence of a variant protease of any of claims 1-15. 24. A process for preparing a fermentation product from starch-containing material, comprising the steps of: (a) liquifying starch-containing material in the presence of an alpha-amylase; (b) saccharifying the liquefied material obtained in step (a) using a glucoamylase; (c) fermenting using a fermenting organism; wherein a variant protease of any of claims 1-15 is present during step b) and/or c). 25. The process of any of claims 23-24, wherein the fermentation product is ethanol and the fermentation organism is Saccharomyces cerevisiae. 26. Host cell according to claim 19, expressing the variants of any of claims 1-15, wherein the host cell is a yeast cell, particularly a Saccharomyces, such as Saccharomyces cerevisiae. 27. The method of any of claims 23-24, wherein the host cell of claim 26 is applied as the fermenting organism in the fermentation step and the fermentation product is ethanol.